Tissue-derived tissuegenic implants, and methods of fabricating and using same

ABSTRACT

The disclosure provides implants containing a plurality of particles containing at least one population of viable osteogenic cells adherent to and resident in an osteoconductive matrix or at least one viable population of osteogenic cells caused to be in contact with the osteoconductive matrix; methods of fabricating the implants; and use of the implants in bone repair. The implant further contains an osteoinductive component. An example of an osteoinductive component is a demineralized bone matrix in the form of particles or fibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/108,856, filed on May 16, 2011, which claims priority fromU.S. Provisional Patent Application Ser. No. 61/345,057, filed May 14,2010, and the disclosures of the aforesaid earlier applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The described invention relates to surgical implants that contain cells,growth factors, and a physical matrix, and methods of fabricationthereof, in particular, to implantable compositions for bone repaircontaining an osteoconductive matrix, endogenous cells, and anosteoinductive matrix.

BACKGROUND OF THE INVENTION 1. Tissue Compartments

In multicellular organisms, cells that are specialized to perform commonfunctions are usually organized into cooperative assemblies embedded ina complex network of secreted extracellular macromolecules, theextracellular matrix (ECM), to form specialized tissue compartments.Individual cells in such tissue compartments are in contact with ECMmacromolecules. The ECM helps hold the cells and compartments togetherand provides an organized lattice or scaffold within which cells canmigrate and interact with one another. In many cases, cells in acompartment can be held in place by direct cell-cell adhesions. Invertebrates, such compartments may be of four major types, a connectivetissue (CT) compartment, an epithelial tissue (ET) compartment, a muscletissue (MT) compartment and a nervous tissue (NT) compartment, which arederived from three embryonic germ layers: ectoderm, mesoderm andendoderm. The NT and portions of the ET compartments are differentiatedfrom the ectoderm; the CT, MT and certain portions of the ETcompartments are derived from the mesoderm; and further portions of theET compartment are derived from the endoderm.

1.1. Extracellular Matrix

The ECM is an intricate network of secreted extracellular macromoleculesthat largely fills the extracellular space in the tissue compartmentsand comprises large polymeric complexes of glycosaminoglycans (GAGs) andproteoglycans. GAGs are negatively charged unbranched polysaccharidechains comprising repeating disaccharide units. Each repeatingdisaccharide unit of a GAG chain contains an amino sugar(N-acetylglucosamine or N-acetyl glucosamine), which in most cases issulfated, and an -uronic acid (glucuronic or iduronic acid). Four maintypes of GAG molecules are distinguished based on sugar residues, typeof linkage, number and location of sulfate groups: (1) hyaluronan; (2)chondroitan sulfate and dermatan sulfate; (3) heparan sulfate andheparin; and (4) keratin sulfate.

GAG chains are inflexible and tend to adopt extended conformationsoccupying a huge volume relative to their mass, forming gels even at lowconcentrations. Their high density of negative charges attracts cations,such as Na⁺, that are effective in osmotic absorption of large amountsof water into the matrix. This creates high turgor enabling the ECM towithstand compressive forces.

Hyaluronan (also termed hyaluronic acid or hyaluronate) (HA), whichcomprises a regular repeating sequence of up to 25,000 nonsulfateddisaccharide units, serves many functions, many of which depend on thebinding of HA-binding proteins and proteoglycans, which are eitherthemselves constituents of the ECM or are integral constituents of cellsurfaces. For example, HA resists compressive forces in joints as amajor constituent of joint fluid serving as a lubricant; serves as aspace filler during embryonic development; creates a cell-free space inepithelial compartment to allow cell migration during the formation ofheart, cornea and other organs; and plays a role in wound repair. ExcessHA is usually degraded by hyaluronidase.

All GAGs, except for HA, are covalently linked to proteins in the formof proteoglycans. During their synthesis, the polypeptide chain ofproteoglycans is synthesized on membrane-bound ribosomes and threadedinto the lumen of endoplasmic reticulum, from which they are sorted inthe Golgi apparatus, and assembled with polysaccharide chains. Whilestill in the Golgi, proteoglycans undergo a series of sequential andcoordinated sulfation and epimerization reactions to produce sulfatedproteoglycans. Sulfated and nonsulfated proteoglycans then travelthrough the Golgi network and are ultimately secreted into the ECM byexocytosis with the help of secretory vesicles.

Proteoglycans are heterogenous molecules, with core proteins ranging inmolecular weight from 10 kD to about 600 kD and with attached GAG chainsvarying in number and type, further modified by a complex variablepattern of sulfate groups. At least one of the proteoglycan sugar sidechains is a GAG; the core protein is usually a glycoprotein, but maycomprise up to 95% carbohydrate by weight, mostly as long unbranched GAGchains up to at least 80 sugar residues long.

Proteoglycans along with their attached GAG chains regulate theactivities of secreted macromolecules. They can serve as selectivemolecular sieves regulating a size-based trafficking of molecules andcells, and play a role in cell-cell signaling. Proteoglycans modulatethe activities of secreted factors, such as growth factors andcytokines, by binding to them For example, binding of fibroblast growthfactor (FGF) to heparan sulfate chains of proteoglycans is required forFGF activation of its cell surface receptors. On the other hand, forexample, binding of a ubiquitous growth regulatory factor, such astransforming growth factor β (TGF-β) to core proteins of several ECMproteoglycans, such as decorin, results in inhibition of TGF-β activity.Proteoglycans also bind and regulate the activities of other types ofsecreted proteins, such as proteases and protease inhibitors.Cell-surface proteoglycans also may act as co-receptors: for example,syndecan binds to FGF and presents it to the FGF-receptor. Similarly,betaglycan binds to TGF-β and presents it to TGF-β receptors.

Collagens and elastin are the major fibrous proteins of the ECM.Collagens comprise a family of highly characteristic fibrous proteinsand are a major component of skin and bone. Collagen fibers consist ofglobular units of the collagen subunit tropocollagen. Each tropocollagensubunit molecule comprises three polypeptide chains, called α chains,each exhibiting a left-handed helical conformation, that are wrappedaround each other in a right-handed coiled coil structure, also called atriple helix or super helix. A characteristic feature of collagen is arepeating tripeptide unit comprising Glycine-Proline-X orGlycine-X-Hydroxyproline, where X may be any amino acid. The presence ofGlycine at every third position in a collagen unit is critical formaintaining the coiled coil structure, since each repeating glycineresidue sits on the interior axis of the helix, which sterically hindersbulkier sidechains. Prolines and hydroxyprolines help stabilize thetriple helix. Collagen is secreted as procollagen molecules, whichundergo proteolytic processing and subsequent assembly to formcollagenous fibrils. Collagens are highly glycosylated during proteintrafficking through intracellular secretory pathways.

Collagens are classified into various types depending on the nature oftheir a chains. Table 1 lists types of collagen, composition, class anddistribution. (Reproduced from Shoulders and Raines, Annu Rev. Biochem.2009, 78: 929-958 and Bailey's Textbook of Microscopic Anatomy, Kelly etal., Williams and Wilkins, 18^(th) edition, 1984).

TABLE 1 Collagen Type, Class and Distribution Collagen Type CompositionClass Distribution I α_(l)[I]₂α₂[I] Fibrillar Dermis, tendon, ligament,bone, cornea II α₁[II]₃ Fibrillar Cartilage, intervertebral disc,vitreous body III α₁[III]₃ Fibrillar Fetal skin, cardiovascular system,basal lamina, intestine. IV α₁[IV]₂α₂[IV]; Network Basal lamina,external lamina α₃[IV]α₄[IV]α₅[IV]; α₅[IV]₂α₆[IV] V α₁[V]₃; FibrillarBone, dermis, cornea, placenta α₁[V]₂α₂[V]; α₁[V]α₂[V]α₃[V] VI α₁[VI]α₂[VI]α₃[VI]; Network Bone, cartilage, cornea, dermis α₁[VI]α₂[VI]α₄[VI]VII α₁[VII]₂α₂[VII] Anchoring fibril Dermis, bladder VIII α₁[VIII]₃;Network Dermis, brain, heart, kidney α₂[VIII]₃; α₁[VIII]₂α₂[VIII] IXα₁[IX]α₂[IX]α₃[IX] FACIT^(a) Cartilage, cornea, vitreous X α₁[X]₃Network Cartilage XI α₁[XI]α₂[XI]α₃[XI] Fibrillar Cartilage,intervertebral disc XII α₁[XII]₃ FACIT Dermis, tendon XIII α₁[XIII]₃MACIT^(a) Endothelial cells, dermis, eye, heart XIV α₁[XIV]₃ FACIT Bone,dermis, cartilage XV MULTIPLEXIN^(a) Capillaries, testis, kidney, heart,bone XVI FACIT Dermis, kidney XVII α₁[XVII]₃ MACIT Hemidesmosomes inepithelia XVIII MULTIPLEXIN Basal lamina, liver XIX FACIT Basal laminaXX FACIT Cornea XXI FACIT Stomach, kidney XXII FACIT Tissue junctionsXXIII MACIT Heart, retina XXIV Fibrillar Bone, cornea XXV MACIT Brain,heart, testis XXVI FACIT Testis, ovary XXVII Dermis, sciatic nerve XXIXDermis ^(a)Abbreviations: FACIT, fibril-associated collagen withinterrupted triple helices; MACIT, membrane-associated collagen withinterrupted triple helices; MULTIPLEXIN, multiple triple helix domains.

A network of elastic fibers in the ECM offers resilience and elasticityso that organs are able to recoil following transient stretch. Elasticfibers primarily comprise the fibrous protein elastin, a highlyhydrophobic protein about 750 amino acids in length that is rich inproline and glycine, is not glycosylated and is low in hydroxyprolineand hyroxylysine. Elastin molecules are secreted into the ECM andassemble into elastic fibers close to the plasma membrane. Uponsecretion, elastin molecules become highly cross-linked to form anextensive network of fibers and sheets.

The ECM also comprises many non-collagen adhesive proteins, usually withmultiple domains containing binding sites of other macromolecules andfor cell-surface receptors. One such adhesive protein, fibronectin, is alarge glycoprotein comprising two subunits joined by a pair of disulfidebonds near the carboxy termini. Each subunit is folded into a series ofrod-like domains interspersed by regions of flexible polypeptide chains.Each domain further comprises repeating modules of various types. Onemajor type of fibronectin repeating module, called type III fibronectinrepeat, is about 90 amino acids in length and occurs at least 15 timesin each subunit. Fibronectin type III repeats have characteristicArg-Gly-Asp (RGD) tripeptide repeats that function as binding sites forother proteins such as collagen, heparin or cell surface receptors.Fibronectin not only plays an important role in cell adhesion to theECM, but also in guiding cell migration in vertebrate embryos.

Laminin, another adhesive glycoprotein of the ECM, is a majorconstituent (along with type IV collagen and another glycoprotein,entactin) of the basal lamina, a tough sheet of ECM formed at the baseof epithelial cells. Laminin is a large flexible complex, about 850 kDin molecular weight, with three very long polypeptide chains arranged inthe form of an asymmetric cross held together with disulfide bonds.Laminin contains numerous functional domains, e.g., one binds to type IVcollagen, one to heparan sulfate, one to entactin and two or more tolaminin receptor proteins on the cell surface.

1.2. Stem Cells

The term “stem cells” as used herein refers to undifferentiated cellshaving high proliferative potential with the ability to self-renew thatcan generate daughter cells that can undergo terminal differentiationinto more than one distinct cell phenotype. Stem cells are distinguishedfrom other cell types by two characteristics. First, they areunspecialized cells capable of renewing themselves through celldivision, sometimes after long periods of inactivity. Second, undercertain physiologic or experimental conditions, they can be induced tobecome tissue- or organ-specific cells with special functions. In someorgans, such as the gut and bone marrow, stem cells regularly divide torepair and replace worn out or damaged tissues. In other organs,however, such as the pancreas and the heart, stem cells only divideunder special conditions.

Embryonic stem cells (EmSC) are stem cells derived from an embryo thatare pluripotent, i.e., they are able to differentiate in vitro intoendodermal, mesodermal and ectodermal cell types.

Adult (somatic) stem cells are undifferentiated cells found amongdifferentiated cells in a tissue or organ. Their primary role in vivo isto maintain and repair the tissue in which they are found. Adult stemcells have been identified in many organs and tissues, including brain,bone marrow, peripheral blood, blood vessels, skeletal muscles, skin,teeth, gastrointestinal tract, liver, ovarian epithelium, and testis.Adult stem cells are thought to reside in a specific area of eachtissue, known as a stem cell niche, where they may remain quiescent(non-dividing) for long periods of time until they are activated by anormal need for more cells to maintain tissue, or by disease or tissueinjury. Examples of adult stem cells include, but not limited to,hematopoietic stem cells, mesenchymal stem cells, neural stem cells,epithelial stem cells, and skin stem cells.

Hematopoietic Stem Cells (HSCs)

Hematopoietic stem cells (also known as the colony-forming unit of themyeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rarepluripotential cells within the blood-forming organs that areresponsible for the continued production of blood cells during life.While there is no single cell surface marker exclusively expressed byhematopoietic stem cells, it generally has been accepted that human HSCshave the following antigenic profile: CD 34+, CD59+, Thy1+(CD90),CD38low/−, C-kit−/low and, lin−. CD45 is also a common marker of HSCs,except platelets and red blood cells. HSCs can generate a variety ofcell types, including erythrocytes, neutrophils, basophils, eosinophils,platelets, mast cells, monocytes, tissue macrophages, osteoclasts, andthe T and B lymphocytes. The regulation of hematopoietic stem cells is acomplex process involving self-renewal, survival and proliferation,lineage commitment and differentiation and is coordinated by diversemechanisms including intrinsic cellular programming and externalstimuli, such as adhesive interactions with the micro-environmentalstroma and the actions of cytokines.

Different paracrine factors are important in causing hematopoietic stemcells to differentiate along particular pathways. Paracrine factorsinvolved in blood cell and lymphocyte formation are called cytokines.Cytokines can be made by several cell types, but they are collected andconcentrated by the extracellular matrix of the stromal (mesenchymal)cells at the sites of hematopoiesis. For example, granulocyte-macrophagecolony-stimulating factor (GM-CSF) and the multilineage growth factorIL-3 both bind to the heparan sulfate glycosaminoglycan of the bonemarrow stroma. The extracellular matrix then presents these factors tothe stem cells in concentrations high enough to bind to their receptors.

Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) (also known as bone marrow stromal stemcells or skeletal stem cells) are non-blood adult stem cells found in avariety of tissues. They are characterized by their spindle-shapemorphologically; by the expression of specific markers on their cellsurface; and by their ability, under appropriate conditions, todifferentiates along a minimum of three lineages (osteogenic,chondrogenic, and adipogenic).

No single marker that definitely delineates MSCs in vivo has beenidentified due to the lack of consensus regarding the MSC phenotype, butit generally is considered that MSCs are positive for cell surfacemarkers CD105, CD166, CD90, and CD44 and that MSCs are negative fortypical hematopoietic antigens, such as CD45, CD34, and CD14. As for thedifferentiation potential of MSCs, studies have reported thatpopulations of bone marrow-derived MSCs have the capacity to developinto terminally differentiated mesenchymal phenotypes both in vitro andin vivo, including bone, cartilage, tendon, muscle, adipose tissue, andhematopoietic-supporting stroma. Studies using transgenic and knockoutmice and human musculoskeletal disorders have reported that MSCdifferentiate into multiple lineages during embryonic development andadult homeostasis.

Analyses of the in vitro differentiation of MSCs under appropriateconditions that recapitulate the in vivo process have led to theidentification of various factors essential for stem cell commitment.Among them, secreted molecules and their receptors (e.g., transforminggrowth factor-β), extracellular matrix molecules (e.g., collagens andproteoglycans), the actin cytoskeleton, and intracellular transcriptionfactors (e.g., Cbfa1/Runx2, PPARγ, Sox9, and MEF2) have been shown toplay important roles in driving the commitment of multipotent MSCs intospecific lineages, and maintaining their differentiated phenotypes.

For example, it has been shown that osteogenesis of MSCs, both in vitroand in vivo, involves multiple steps and the expression of variousregulatory factors. During osteogenesis, multipotent MSCs undergoasymmetric division and generate osteoprecursors, which then progress toform osteoprogenitors, preosteoblasts, functional osteoblasts, andeventually osteocytes. This progression from one differentiation stageto the next is accompanied by the activation and subsequent inactivationof transcription factors, i.e., Cbfa1/Runx2, Msx2, Dlx5, Osx, andexpression of bone-related marker genes, i.e., osteopontin, collagentype I, alkaline phosphatase, bone sialoprotein, and osteocalcin.

Members of the Wnt family also have been shown to impact MSCosteogenesis. Wnts are a family of secreted cysteine-rich glycoproteinsthat have been implicated in the regulation of stem cell maintenance,proliferation, and differentiation during embryonic development.Canonical Wnt signaling increases the stability of cytoplasmic β-cateninby receptor-mediated inactivation of GSK-3 kinase activity and promotesβ-catenin translocation into the nucleus. The active β-catenin/TCF/LEFcomplex then regulates the transcription of genes involved in cellproliferation. In humans, mutations in the Wnt co-receptor, LRP5, leadto defective bone formation. “Gain of function” mutation results in highbone mass, whereas “loss of function” causes an overall loss of bonemass and strength, indicating that Wnt signaling is positively involvedin embryonic osteogenesis. Canonical Wnt signaling pathway alsofunctions as a stem cell mitogen via stabilization of intracellularβ-catenin and activation of the β-catenin/TCF/LEF transcription complex,resulting in activated expression of cell cycle regulatory genes, suchas Myc, cyclin D1, and Msx1. When MSCs are exposed to Wnt3a, aprototypic canonical Wnt signal, under standard growth mediumconditions, they show markedly increased cell proliferation and adecrease in apoptosis, consistent with the mitogenic role of Wnts inhematopoietic stem cells. However, exposure of MSCs to Wnt3a conditionedmedium or overexpression of ectopic Wnt3a during osteogenicdifferentiation inhibits osteogenesis in vitro through β-cateninmediated down-regulation of TCF activity. The expression of severalosteoblast specific genes, e.g., alkaline phosphatase, bonesialoprotein, and osteocalcin, is dramatically reduced, while theexpression of Cbfa1/Runx2, an early osteo-inductive transcription factoris not altered, implying that Wnt3a-mediated canonical signaling pathwayis necessary, but not sufficient, to completely block MSC osteogenesis.On the other hand, Wnt5a, a typical non-canonical Wnt member, has beenshown to promote osteogenesis in vitro. Since Wnt3a promotes MSCproliferation during early osteogenesis, it is thought likely thatcanonical Wnt signaling functions in the initiation of early osteogeniccommitment by increasing the number of osteoprecursors in the stem cellcompartment, while non-canonical Wnt drives the progression ofosteoprecursors to mature functional osteoblasts.

Epithelial Stem Cells.

An epithelial membrane is a continuous multicellular sheet composed ofan epithelium adhered to underlying connective tissue. Epithelialmembranes can be cutaneous (e.g. skin), mucous (e.g., gastrointestinallining) and serous (e.g. pleural lining, pericardial lining andperitoneal lining).

Epithelial stem cells line the gastrointestinal tract in deep crypts andgive rise to absorptive cells, goblet cells, paneth cells, andenteroendocrine cells.

Components of the Human Gastrointestinal Tract

The gastrointestinal tract is a continuous tube that extends from themouth to the anus. On a gross level, the gastrointestinal tract iscomposed of the following organs: the mouth, most of the pharynx, theesophagus, the stomach, the small intestine (duodenum, jejunum andileum), and the large intestine. Each segment of the gastrointestinaltract participates in the absorptive processes essential to digestion byproducing chemical substances that facilitate digestion of orally takenfoods, liquids, and other substances such as therapeutic agents.

Within the gastrointestinal tract, the small intestine, the site of mostdigestion and absorption, is structured specifically for these importantfunctions. The small intestine is divided into three segments: theduodenum, the jejunum, and the ileum. The absorptive cells of the smallintestine produce several digestive enzymes called the ‘brush-border’enzymes. Together with pancreatic and intestinal juices, these enzymesfacilitate the absorption of substances from the chime in the smallintestine. The large intestine, the terminal portion of thegastrointestinal tract, contributes to the completion of absorption, theproduction of certain vitamins, and the formation and expulsion offeces.

At the cellular level, the epithelium is a purely cellular avasculartissue layer that covers all free surfaces (cutaneous, mucous, andserous) of the body including the glands and other structures derivedfrom it. It lines both the exterior of the body, as skin, and theinterior cavities and lumen of the body. While the outermost layer ofhuman skin is composed of dead stratified squamous, keratinizedepithelial cells, mucous membranes lining the inside of the mouth, theesophagus, and parts of the rectum are themselves lined bynonkeratinized stratified squamous epithelium. Epithelial cell lines arepresent inside of the lungs, the gastrointestinal tract, and thereproductive and urinary tracts, and form the exocrine and endocrineglands.

Epithelial cells are involved in secretion, absorption, protection,transcellular transport, sensation detection and selective permeability.There are variations in the cellular structures and functions in theepithelium throughout the gastrointestinal tract. The epithelium in themouth, pharynx, esophagus and anal canal is mainly a protective,nonkeratinized, squamous epithelium. The epithelium of the stomach iscomposed of (i) simple columnar cells that participate in nutrient andfluid absorption and secretion, (ii) mucus producing goblet cells thatparticipate in protective and mechanical functions, and (iii)enteroendocrine cells that participate in the secretion ofgastrointestinal hormones. Additionally, within the intestine, theepithelial lining provides an important defense barrier againstmicrobial pathogens.

The development of intestinal epithelium involves three major phases: 1)an early phase of epithelial proliferation and morphogenesis; 2) anintermediate period of cellular differentiation in which the distinctivecell types characteristic of intestinal epithelium appear; and 3) afinal phase of biochemical and functional maturation. Intestinal crypts,located at the base of villi, contain stem cells which supply the entireepithelial cell surface with a variety of epithelial cell subtypes.These specialized cells provide for an external environment-internalenvironment interface, ion and fluid secretion and reabsorption, antigenrecognition, hormone secretion, and surface protection. The exposure ofepithelial cells on the surfaces of the intestinal lumen subjects themto a wide range of assaults, including microbial, chemical, and physicalforces; thus they also may contribute to patho-physiologic impairment indiseases. Additionally, these cells are targets for inflammation,infection, and malignant transformation.

Within the intestinal tract, the epithelium forms upon stem celldifferentiation.

Molecular Markers of Gastrointestinal Epithelial Stem Cells

As disclosed in U.S. Published Application No. 2009/0269769, which isincorporated herein by reference in its entirety, there are nouniversally accepted molecular markers that identify gastrointestinalstem cells. However, several markers have been used to identify stemcells in small and large intestinal tissues. These include:β-1-integrin, mushashi-1, CD45, and cytokeratin.

CD45, also called the common leukocyte antigen, T220 and B220 in mice,is a transmembrane protein with cytoplasmic protein tyrosine phosphatase(PTP) activity. CD45 is found in hematopoietic cells except erythrocytesand platelets. CD45 has several isoforms that can be seen in the variousstages of differentiation of normal hematopoietic cells.

Mushashi-1 is an early developmental antigenic marker of stem cells andglial/neuronal cell precursor cells.

β-1-integrin (CD29, fibronectin receptor), is a β-subunit of aheterodimer protein member of the integrin family of proteins; integrinsare membrane receptors involved in cell adhesion and recognition.

Cytokeratins are intermediate filament proteins found in theintracytoplasmic cystoskeleton of the cells that comprise epithelialtissue.

There are four main epithelial cell lineages: (i) columnar epithelialcells, (ii) goblet cells, (iii) enteroendocrine chromaffin cells, and(iv) Paneth cells. Several molecular markers have been used to identifyeach of these lineages.

The markers used to identify columnar epithelial cells include:intestinal alkaline phosphatase (ALP1), sucrase isomaltase (SI),sodium/glucose cotransporter (SLGT1), dipeptidyl-peptidase 4 (DPP4), andCD26. Intestinal alkaline phosphatase (E.C. 3.1.3.1) is a membrane-boundenzyme localized in the brush border of enterocytes in the humanintestinal epithelium. Sucrase-isomaltase (SI, EC 3.2.1.48) is anenterocyte-specific small intestine brush-border membranedisaccharidase. Dipeptidyl-peptidase 4 (E.C. 3.4.14.5) is a membranebound serine-type peptidase. Sodium/glucose transporter (SGLT) mediatestransport of glucose into epithelial cells. SGLT belongs to thesodium/glucose cotransporter family SLCA5. Two different SGLT isoforms,SGLT1 and SGLT2, mediate renal tubular glucose reabsorption in humans.Both of them are characterized by their different substrate affinity.SGLT1 transports glucose as well as galactose, and is expressed both inthe kidney and in the intestine. SGLT2 transports glucose and isbelieved to be responsible for 98% of glucose reabsorption; SGLT2 isgenerally found in the S1 and S2 segments of the proximal tubule of thenephron. CD26 is a multifunctional protein of 110 KDa strongly expressedon epithelial cells (kidney proximal tubules, intestine, and bile duct)and on several types of endothelial cells and fibroblasts and onleukocyte subsets.

The markers used to identify goblet cells include mucin 2 (MUC2) andtrefoil factor 3 (TFF3). Mucin-2, a secreted gel-forming mucin, is themajor gel-forming mucin secreted by goblet cells of the small and largeintestines and is the main structural component of the mucus gel.Intestinal trefoil factor 3 is a nonmucin protein and a product of fullydifferentiated goblet cells.

The markers used to identify enteroendocrine chromaffin cells includechromogranin A (CHGA) and synaptophysin (SYP). Chromogranin A (CHGA) andits derived peptides, which are stored and released from dense-coresecretory granules of neuroendocrine cells, have been implicated asplaying multiple roles in the endocrine, cardiovascular, and nervoussystems. Synaptophysin I (SYP) is a synaptic vesicle membrane proteinthat is ubiquitously expressed throughout the brain without a definitesynaptic function.

The markers used to identify Paneth cells include lysozyme (LYZ),defensin (DEFA1), and matrix metallopeptidase 7 (MMP7). Lysozyme (LYZ ormuramidase) (E.C. 3.2.1.17) catalyzes the hydrolysis of1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins. Defensins (DEFA1) aresmall peptides that are produced by leukocytes and epithelial cells.Human defensin α-1 is a 3.5-kDa, 30-amino-acid peptide that has showneffector functions in host innate immunity against some microorganisms.Matrix metalloproteinases (MMPs) are a family of metal-dependant enzymesthat are responsible for the degradation of extracellular matrixcomponents. MMPs are involved in various physiologic processes, such asembryogenesis and tissue remodeling and also play a role in invasion andmetastasis of tumor cells, which require proteolysis of basal membranesand extracellular matrix.

Neural Stem Cells

The adult mammalian brain contains multipotent neural stem cells (NSCs)that have the capacity to self-renew and are responsible forneurogenesis and maintenance of specific regions of the adult brain.Neural stem cells can generate astrocytes, oligodendrocytes, andneurons. Self-renewal and differentiation of neural stem cells aredirected by interactions within a complex network of intrinsicregulators and extrinsic factors. Recent proteomic analyses haveidentified a horde of transcription factors belonging to theWnt/β-catenin, Notch and Sonic Hedgehog (shh) pathways, in addition toepigenetic modifications, microRNA networks and extrinsic growth factornetworks, including but not limited to the FGFs and BMPs. (Yun ey al.,2010, J. Cell. Physiol. 225: 337-347).

With the advent of high throughput microarray and proteomictechnologies, a number of different molecular signatures of neural stemcells have been identified, including but not limited to CD133/promini,nestin, NCAM, the HMG-box transcription factor, Sox2 and the bHLHprotein, Olig2. (Holmberg et al., 2011, PLoS One., 6(3): e18454;Hombach-Klonisch et al., 2008, J. Mol. Med. 86(12): 1301-1314).

Skin Stem Cells.

Several different adult stem cell populations with distinct molecularsignatures are responsible for maintaining skin homeostasis. Theseinclude, but are not limited to, epidermal stem cells of theinterfollicular region, epidermal stem cells of the hair follicle (alsoknown as the bulge stem cells), dermal stem cells, dermal papilla stemcells, and sebaceous gland stems. The epidermal stem cells areectodermal in origin while the dermal stem cells originate from themesoderm and are mesenchymal in nature. (Zouboulis et al., 2008, Exp.Gerontol., 43: 986-997).

The interfollicular epidermal stem cells reside in the basal layer ofthe epidermis and give rise to keratinocytes, which migrate to thesurface of the skin and form a protective layer. A diverse range ofmolecular signatures has been described for such epidermal stem cellsincluding but not limited to high α6-integrin, low CD71, high Delta 1(Notch signaling ligand) and high CD200 expression levels. Thefollicular stem cells located at the base of hair follicles give rise toboth hair follicle and to the epidermis. These are characterized byCytokeratin 15 (K15) immunostaining and high levels of β1-integrin.Dermal stem cell marker proteins include but are not limited to nestin,fibronectin and vimentin, the surface markers for dermal papilla stemcells include mesenchymal stem cell markers such as for example CD44,CD73 and CD90 and sebaceous stem cells express keratin 14. (Zouboulis etal., 2008, Exp. Gerontol., 43: 986-997).

In addition, adult somatic cells can be reprogrammed to enter anembryonic stem cell-like state by being forced to express a set oftranscription factors, for example, Oct-3/4 (or Pou5f1, the Octamertranscription factor-3/4), the Sox family of transcription factors(e.g., Sox-1, Sox-2, Sox-3, and Sox-15), the Klf family transcriptionfactors (Klf-1, Klf-2, Klf-4, and Klf-5), and the Myc family oftranscription factors (e.g., c-Myc, N-Myc, and L-Myc). For example,human inducible Pluripotent Stem cells (iPSCs) are cells reprogrammed toexpress transcription factors that express stem cell markers and arecapable of generating cells characteristic of all three germ layers(i.e., ectoderm, mesoderm, and endoderm).

1.3. Stem Cell Niches

Adult tissue compartments contain endogenous niches of adult stem cellsthat are capable of differentiating into diverse cell lineages ofdetermined endodermal, mesodermal or ectodermal fate depending on theirlocation in the body. For example, in the presence of an appropriate setof internal and external signals, bone marrow-derived adulthematopoietic stem cells (HSCs) have the potential to differentiate intoblood, endothelial, hepatic and muscle cells; brain-derived neural stemcells (NSCs) have the potential to differentiate into neurons,astrocytes, oligodendrocytes and blood cells; gut- and epidermis-derivedadult epithelial stem cells (EpSCs) have the potential to give rise tocells of the epithelial crypts and epidermal layers; adipose-derivedstem cells (ASCs) have the potential to give rise to fat, muscle,cartilage, endothelial cells, neuron-like cells and osteoblasts; andbone-marrow-derived adult mesenchymal stem cells (MSCs) have thepotential to give rise to bone, cartilage, tendon, adipose, muscle,marrow stroma and neural cells.

Endogenous adult stem cells are embedded within the ECM component of agiven tissue compartment, which, along with support cells, form thecellular niche. Such cellular niches within the ECM scaffold togetherwith the surrounding microenvironment contribute important biochemicaland physical signals, including growth factors and transcription factorsrequired to initiate stem cell differentiation into committed precursorscells and subsequent precursor cell maturation to form adult tissuecells with specialized phenotypic and functional characteristics.

1.4. Growth Factors

Growth factors are extracellular polypeptide molecules that bind to acell-surface receptor triggering an intracellular signaling pathway,leading to proliferation, differentiation, or other cellular response.These pathways stimulate the accumulation of proteins and othermacromolecules, and they do so by both increasing their rate ofsynthesis and decreasing their rate of degradation. One intracellularsignaling pathway activated by growth factor receptors involves theenzyme PI 3-kinase, which adds a phosphate from ATP to the 3 position ofinositol phospholipids in the plasma membrane. The activation of PI3-kinase leads to the activation of several protein kinases, includingS6 kinase. The S6 kinase phosphorylates ribosomal protein S6, increasingthe ability of ribosomes to translate a subset of mRNAs, most of whichencode ribosomal components, as a result of which, protein synthesisincreases. When the gene encoding S6 kinase is inactivated inDrosophila, cell numbers are normal, but cell size is abnormally small,and the mutant flies are small. Growth factors also activate atranslation initiation factor called eIF4E, further increasing proteinsynthesis and cell growth.

Growth factor stimulation also leads to increased production of the generegulatory protein Myc, which plays a part in signaling by mitogens. Mycincreases the transcription of a number of genes that encode proteinsinvolved in cell metabolism and macromolecular synthesis. In this way,it stimulates both cell metabolism and cell growth.

Some extracellular signal proteins, including platelet-derived growthfactor (PDGF), can act as both growth factors and mitogens, stimulatingboth cell growth and cell-cycle progression. This functional overlap isachieved in part by overlaps in the intracellular signaling pathwaysthat control these two processes. The signaling protein Ras, forexample, is activated by both growth factors and mitogens. It canstimulate the PI3-kinase pathway to promote cell growth and theMAP-kinase pathway to trigger cell-cycle progression. Similarly, Mycstimulates both cell growth and cell-cycle progression. Extracellularfactors that act as both growth factors and mitogens help ensure thatcells maintain their appropriate size as they proliferate.

Since many mitogens, growth factors, and survival factors are positiveregulators of cell-cycle progression, cell growth, and cell survival,they tend to increase the size of organs and organisms. In some tissues,however, cell and tissue size also is influenced by inhibitoryextracellular signal proteins that oppose the positive regulators andthereby inhibit organ growth. The best-understood inhibitory signalproteins are TGF-β and its relatives. TGF-β inhibits the proliferationof several cell types, either by blocking cell-cycle progression in G1or by stimulating apoptosis. TGF-β binds to cell-surface receptors andinitiates an intracellular signaling pathway that leads to changes inthe activities of gene regulatory proteins called Smads. This results incomplex changes in the transcription of genes encoding regulators ofcell division and cell death.

Bone morphogenetic protein (BMP), a TGF-β family member, helps triggerthe apoptosis that removes the tissue between the developing digits inthe mouse paw. Like TGF-β, BMP stimulates changes in the transcriptionof genes that regulate cell death.

Fibroblast Growth Factor (FGF)

The fibroblast growth factor (FGF) family currently has over a dozenstructurally related members. FGF1 is also known as acidic FGF; FGF2 issometimes called basic FGF (bFGF); and FGF7 sometimes goes by the namekeratinocyte growth factor. Over a dozen distinct FGF genes are known invertebrates; they can generate hundreds of protein isoforms by varyingtheir RNA splicing or initiation codons in different tissues. FGFs canactivate a set of receptor tyrosine kinases called the fibroblast growthfactor receptors (FGFRs). Receptor tyrosine kinases are proteins thatextend through the cell membrane. The portion of the protein that bindsthe paracrine factor is on the extracellular side, while a dormanttyrosine kinase (i.e., a protein that can phosphorylate another proteinby splitting ATP) is on the intracellular side. When the FGF receptorbinds an FGF (and only when it binds an FGF), the dormant kinase isactivated, and phosphorylates certain proteins within the respondingcell, activating those proteins.

FGFs are associated with several developmental functions, includingangiogenesis (blood vessel formation), mesoderm formation, and axonextension. While FGFs often can substitute for one another, theirexpression patterns give them separate functions. FGF2 is especiallyimportant in angiogenesis, whereas FGF8 is involved in the developmentof the midbrain and limbs.

The expression levels of angiogenic factors, such as VEGF, IGF, PDGF,HGF, FGF, TGFm Angiopoeitin-1, and stem cell factor (SCF) have beenfound to differ amongst bone-derived-, cartilage-derived-, andadipose-derived MSCs. (Peng et al., 2008, Stems Cells and Development,17: 761-774).

Insulin-Like Growth Factor (IGF-1)

IGF-1, a hormone similar in molecular structure to insulin, hasgrowth-promoting effects on almost every cell in the body, especiallyskeletal muscle, cartilage, bone, liver, kidney, nerves, skin,hematopoietic cell, and lungs. It plays an important role in childhoodgrowth and continues to have anabolic effects in adults. IGF-1 isproduced primarily by the liver as an endocrine hormone as well as intarget tissues in a paracrine/autocrine fashion. Production isstimulated by growth hormone (GH) and can be retarded by undernutrition,growth hormone insensitivity, lack of growth hormone receptors, orfailures of the downstream signaling molecules, including SHP2 andSTAT5B. Its primary action is mediated by binding to its specificreceptor, the Insulin-like growth factor 1 receptor (IGF1R), present onmany cell types in many tissues. Binding to the IGF1R, a receptortyrosine kinase, initiates intracellular signaling; IGF-1 is one of themost potent natural activators of the AKT signaling pathway, astimulator of cell growth and proliferation, and a potent inhibitor ofprogrammed cell death. IGF-1 is a primary mediator of the effects ofgrowth hormone (GH). Growth hormone is made in the pituitary gland,released into the blood stream, and then stimulates the liver to produceIGF-1. IGF-1 then stimulates systemic body growth. In addition to itsinsulin-like effects, IGF-1 also can regulate cell growth anddevelopment, especially in nerve cells, as well as cellular DNAsynthesis.

Transforming Growth Factor Beta (TGF-β)

There are over 30 structurally related members of the TGF-β superfamily,and they regulate some of the most important interactions indevelopment. The proteins encoded by TGF-β superfamily genes areprocessed such that the carboxy-terminal region contains the maturepeptide. These peptides are dimerized into homodimers (with themselves)or heterodimers (with other TGF-β peptides) and are secreted from thecell. The TGF-β superfamily includes the TGF-β family, the activinfamily, the bone morphogenetic proteins (BMPs), the Vg-1 family, andother proteins, including glial-derived neurotrophic factor (GDNF,necessary for kidney and enteric neuron differentiation) and Müllerianinhibitory factor, which is involved in mammalian sex determination.TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating theformation of the extracellular matrix between cells and for regulatingcell division (both positively and negatively). TGF-β1 increases theamount of extracellular matrix epithelial cells make both by stimulatingcollagen and fibronectin synthesis and by inhibiting matrix degradation.TGF-βs may be critical in controlling where and when epithelia canbranch to form the ducts of kidneys, lungs, and salivary glands.

The members of the BMP family were originally discovered by theirability to induce bone formation. Bone formation, however, is only oneof their many functions, and they have been found to regulate celldivision, apoptosis (programmed cell death), cell migration, anddifferentiation. BMPs can be distinguished from other members of theTGF-β superfamily by their having seven, rather than nine, conservedcysteines in the mature polypeptide. The BMPs include proteins such asNodal (responsible for left-right axis formation) and BMP4 (important inneural tube polarity, eye development, and cell death).

Neural Epidermal Growth-Factor-Like 1 (NELL1)

Neural epidermal growth-factor-like 1 (NEL-like 1, NELL1) is a gene thatencodes an 810-amino acid polypeptide, which trimerizes to form a matureprotein involved in the regulation of cell growth and differentiation.The neural epidermal growth-factor-like (nel) gene first was detected inneural tissue from an embryonic chicken cDNA library, and its humanorthologue NELL1 was discovered later in B-cells. Studies have reportedthe presence of NELL in various fetal and adult organs, including, butnot limited to, the brain, kidneys, colon, thymus, lung, and smallintestine.

NELL1—General Structure

Generally, the arrangement of the functional domains of the 810 aminoacid NELL1 protein bears resemblance to thrombospondin-1 (“THBS1”) andconsists of a thrombospondin N-terminal domain (“TSPN”) and several vonWillebrand factor, type C (“VWC”), and epidermal growth-factor (“EGF”)domains.

Additional studies have shown that there are two transcript variantsencoding different isoforms. The nel-like 1 isoform 1 precursortranscript variant represents the longer transcript and encodes thelonger isoform 1.

The conserved domains of the nel-like 1 isoform 1 precursor transcriptreside in seven regions of the isoform 1 peptide and include: (1) a TSPNdomain/Laminin G superfamily domain; (2) a VWC domain; (3) an EGF-likedomain; (4) an EGF-like domain; (5) an EGF-like domain; (6) an EGF-likedomain and (7) a VWC domain.

The first conserved domain region comprises amino acids (amino acids 29to 213) that are most similar to a thrombospondin N-terminal-likedomain. Thrombospondins are a family of related, adhesive glycoproteins,which are synthesized, secreted and incorporated into the extracellularmatrix of a variety of cells, including alpha granules of plateletsfollowing thrombin activation and endothelial cells. They interact witha number of blood coagulation factors and anticoagulant factors, and areinvolved in cell adhesion, platelet aggregation, cell proliferation,angiogenesis, tumor metastasis, vascular smooth muscle growth and tissuerepair. The first conserved domain also comprises amino acids (aminoacids 82 to 206; amino acids 98 to 209) that are similar to a LamininG-like domain. Laminin G-like (LamG) domains usually are Ca²⁺ mediatedreceptors that can have binding sites for steroids, β1-integrins,heparin, sulfatides, fibulin-1, and α-dystroglycans. Proteins thatcontain LamG domains serve a variety of purposes, including signaltransduction via cell-surface steroid receptors, adhesion, migration anddifferentiation through mediation of cell adhesion molecules.

Much of what is known about NELL1 concerns its role in bone development.See, e.g., U.S. Pat. No. 7,884,066, U.S. Pat. No. 7,833,968, U.S. Pat.No. 7,807,787, U.S. Pat. No. 7,776,361, U.S. Pat. No. 7,691,607, U.S.Pat. No. 7,687,462, U.S. Pat. No. 7,544,486, and U.S. Pat. No.7,052,856, the entire contents of which are incorporated herein byreference. It generally is believed that during osteogenicdifferentiation, NELL1 signaling may involve an integrin-relatedmolecule and tyrosine kinases that are triggered by NELL1 binding to aNELL1 specific receptor and a subsequent formation of an extracellularcomplex. As thus far understood, in human NELL1 (hNELL1), the laminin Gdomain comprises about 128 amino acid residues that show a high degreeof similarity to the laminin G domain of extracellular matrix (“ECM”)proteins, such as human laminin α3 chain (hLAMA3), mouse laminin α3chain (mLAMA3), human collagen 11 α3 chain (hCOLA1), and humanthrombospondin-1 (hTSP1). This complex facilitates either activation ofTyr-kinases, inactivation of Tyr phosphatases, or intracellularrecruitment of Tyr-phosphorylated proteins. The ligand bound integrin(cell surface receptors that interact with ECM proteins such as, forexample, laminin 5, fibronectin, vitronectin, TSP1/2) transduces thesignals through activation of the focal adhesion kinase (FAK) followedby indirect activation of the Ras-MAPK cascade, and then leads toosteogenic differentiation through Runx2; the laminin G domain isbelieved to play a role in the interaction between integrins and a 67kDa laminin receptor.

The second conserved domain (amino acids 273 to 331) and seventhconserved domain (amino acids 701 to 749; amino acids 703 to 749) aresimilar to von Willebrand factor type C (VWC) domains, also known aschordin-like repeats. VWC domains occur in numerous proteins of diversefunctions. It is thought that these domains may be involved in proteinoligomerization.

The third conserved domain (amino acids 434 to 471; amino acids 434 to466), fourth conserved domain (amino acids 478 to 512), fifth conserveddomain (amino acids 549 to 586; amino acids 549 to 582), and sixthconserved domain (amino acids 596 to 627; amino acids 596 to 634) aresimilar to a calcium-binding EGF-like domain. Calcium-binding EGF-likedomains are present in a large number of membrane-bound andextracellular (mostly animal) proteins. Many of these proteins requirecalcium for their biological function. Calcium-binding sites have beenfound to be located at the N-terminus of particular EGF-like domains,suggesting that calcium-binding may be crucial for numerousprotein-protein interactions. Six conserved core cysteines form threedisulfide bridges as in non-calcium-binding EGF domains whose structuresare very similar.

The nel-like 1 isoform 2 precursor transcript variant lacks an alternatein-frame exon compared to variant 1. The resulting isoform 2, which hasthe same N- and C-termini as isoform 1 but is shorter compared toisoform 1, has six conserved regions including a TSPN domain/LamGsuperfamily domain (amino acids 29 to 313); VWC domains (amino acids 273to 331; amino acids 654 to 702); and calcium-binding EGF-like domains(amino acids 478 to 512; amino acids 434 to 471; amino acids 549 to580).

NELL1 and its orthologs are found across several species including Homosapiens (man), Mus musculus (mouse), Rattus norvegicus (rat), Pantroglodytes (chimpanzee), Xenopus (Silurana) tropicalis (frog), Canislupus familiaris (dog), Culex quinquefasciatus (mosquito) Pediculushumanus corporis (head louse), Aedes aegypti (mosquito), Ixodesscapularis (tick), Strongylocentrotus purpuratus (purple sea urchin),and Acyrthosiphon pisum (pea aphid).

NELL1 is Variable

NELL1 comprises several regions susceptible to increased recombination.Studies have indicated that susceptibilities to certain diseases may beassociated with genetic variations within these regions, suggesting theexistence of more than one causal variant in the NELL1 gene. Forexample, in patients suffering irritable bowel syndrome (“IBS”), sixdifferent single nucleotide polymorphisms (SNPs) within NELL1 have beenidentified, with most of these SNPs near the 5′ end of the gene andfewer at the 3′ end. These include R136S and A153T (which reside in theTSPN) and R354W (which resides in a VWC domain). Additional studies haveidentified at least 26 variants comprising some of at least 263 SNPswithin the NELL1 region.

NELL1-Function

The NELL1 protein is a secreted cytoplasmic heterotrimeric protein. Thecomplete role NELL1 plays in vivo remains unknown.

Several studies have indicated that NELL1 may play a role in boneformation, inflammatory bowel disease, and esophageal adenocarcinoma,among others.

NELL1 in Osteogenesis

It generally is believed that NELL1 induces osteogenic differentiationand bone formation of osteoblastic cells during development. Studieshave shown that the NELL1 protein (1) transiently activates themitogen-activated protein kinase (“MAPK”) signaling cascade (which isinvolved in various cellular activities such as gene expression,mitosis, differentiation, proliferation and apotosis); and (2) inducesphosphorylation of Runx2 (a transcription factor associated withosteoblast differentiation). Consequently, it generally is believed thatupon binding to a specific receptor, NELL1 transduces an osteogenicsignal through activation of certain Tyr-kinases associated with theRas-MAPK cascade, which ultimately leads to osteogenic differentiation.Studies have shown that bone development is severely disturbed intransgenic mice where over-expression of NELL1 has been shown to lead tocraniosynotosis (premature ossification of the skull and closure of thesutures) and NELL1 deficiency manifests in skeletal defects due toreduced chondrogenesis and osteogenesis.

Additional studies have supported a role for NELL-1 as acraniosynostosis-related gene. For example, three regions within theNELL-1 promoter have been identified that are directly bound andtransactivated by Runx2. Further, studies in rat skullcaps haveindicated that forced expression of Runx2 induces NELL-1 expression(which is suggestive that Nell-1 is a downstream target of Runx2).

2. Cells of the Connective Tissue Compartment

The connective tissue compartment contains cells that primarily functionto elaborate and maintain ECM structure. The character of theextracellular matrix is region-specific and is determined by the amountof the extracellular materials.

Common cell types of connective tissue compartments include:fibroblasts, macrophages, mast cells, and plasma cells. Specializedconnective tissue compartments, such as cartilage, bone, and thevasculature, and those with special properties, such as adipose,tendons, ligaments, etc., have specialized cells to perform specializedfunctions.

2.1. Adipose Tissue Compartment

Adipose tissue compartments are dynamic, multifunctional, ubiquitous andloose connective tissue compartments. Adipose comprises fibroblasts,smooth muscle cells, endothelial cells, leukocytes, macrophages, andclosely packed mature lipid-filled fat cells, termed adipocytes, withcharacteristic nuclei pushed to one side, embedded within an areolarmatrix that are located in subcutaneous layers of skin and muscle(panniculus adiposus), in the kidney region, cornea, breasts,mesenteries, mediastinium, and in the cervical, axillary and inguinalregions. Adipocytes play a primary role in energy storage and inproviding insulation and protection. As sites of energy storage,adipocytes regulate the accumulation or mobilization of triacylglycerolin response to the body's energy requirements and store energy in theform of a single fat droplet of triglycerides.

Adipocyte Matrix

Each adipocyte is surrounded by a thick ECM called the basal lamina. Thestrong adipocyte ECM scaffold lowers mechanical stress by spreadingforces over a large surface area of the adipose tissue compartments. TheECM composition of adipocytes is similar to that of other cell types,but it is the relative quantity of individual components that impartcell specificity. Adipocyte ECM is particularly enriched in collagen VI,a coiled coil comprising α1(VI), α2(VI) and α3(VI) subunits. Collagen VIbinds to collagen IV and also to other matrix proteins such asproteoglycans and fibronectin. Table 2 lists core proteins that havebeen annotated to the adipocyte ECM with current proteomic techniques.(Mariman et al., 2010, Cell. Mol. Life Sci., 67:1277-1292).

TABLE 2 Core Proteins of Human Adipocyte ECM Protein Symbol Basementmembrane-specific heparan sulfate HSPG2 proteoglycan core protein (HSPG)(perlecan) Calreticulin CALR Chitinase-3-like protein 1 CHI311 Coiledcoil domain containing protein 80 CCDC80 Collagen α 1(I) chain COL1A1Collagen α 2(I) chain COL1A2 Collagen α 1(III) chain COL2A1 Collagen α2(IV) chain COL4A2 Collagen α 1(V) chain COL5A1 Collagen α 1(VI) chainCOL6A1 Collagen α 2(VI) chain COL6A2 Collagen α 3(VI) chain COL6A3Collagen α 1(XII) chain COL12A1 Collagen α 1(XIV) chain (undulin)COL14A1 Collagen α 1(XV) chain COL15A1 Collagen α 1(XVIII) chain COL18A1Decorin (bone proteoglycan II) DCN Dermatopontin (tyrosine-rich acidicmatrix DPT protein; early quiescence protein 1) Elastin microfibrilinterface-located protein 1 EMILIN1 Fibronectin (FN) (cold-insolubleglobulin) FN1 Fibulin-1 FBLN1 Fibulin-3 (EGF-containing fibulin-likeFBLN3 extracellular matrix protein 1) Fibulin-5 (developmental arteriesand FBLN5 neural crest EGF-like protein Galectin-1 LGALS1Galectin-3-binding protein (lectin LGALS3BP galactoside-binding soluble3-binding protein) Glypican 1 GPC1 Laminin α-4 chain LAMA4 Laminin β-1chain LAMB1 Laminin β-2 chain LAMB2 Laminin γ-1 chain LAMC1 Lumican(keratan sulfate proteoglycan lumican) LUM Matrilin-2 MATN2Microfibril-associated glycoprotein 4 MFAP4 Mimecan (osteoglycin) OGNNidogen 1 (entactin) NID1 Nidogen 2 (osteonidogen)) NID2 Periostin POSTNProteoglycan 4 PRG4 SPARC (osteonectin) SPARC Spondin-1 (F-spondin)(vascular smooth SPON1 muscle cell growth-promoting factor) Spondin-2(mindin) SPON2 Tenascin-C (TN) (hexabrachion) TNC (cytotactin)(neuronectin) (GMEM) Tenascin-X TNXB Thrombospondin-1 THBS1Thrombospondin-2 THBS2 Transforming growth factor-b- TGFB1 inducedprotein IG-H3 (bIG-H3) Versican core protein (large fibroblast CSPG2proteoglycan) Versican V3 isoform VCAN

Adipocyte ECM undergoes biphasic development during adipogenesis, theprocess of formation of mature adipose tissue compartments. There is aninitial decrease in collagen I and III, whereas their levels come backto pre differentiation state at later stages. Mature adipocyte ECM ismaintained in a dynamic state with constant turnover of ECM componentsby a balance of activities of ECM constructive enzymes and ECMdegradation enzymes. In early stages of differentiation, the balance isshifted towards the constructive factors. (Mariman et al., 2010, Cell.Mol. Life Sci., 67:1277-1292). Maturation of newly synthesized ECMcomponents is initiated in the ER lumen where ECM proteins undergobiochemical modifications and proteolytic processing prior to assembly.For collagen, such modifications include proline- andlysine-hydroxylation and glycosylation and clipping of N- and C-terminalpeptides by respective procollagen-N- and -C-collagenase. Processedproteins are then assembled and secreted into the extracellularenvironment where they undergo further processing by secretedextracellular modification and processing enzymes. As the preadipocytesdifferentiate and begin to store fat, ECM assumes a basal laminarstructure.

Adipose-Derived Stem Cells

Adipose also comprises a population of pluripotent stem cells that havethe potential to give rise to cells of all three embryonic lineages:ectodermal, mesodermal and endodermal. Adipogenesis, which comprises thesteps of differentiation of such pluripotent cells to mature adipocytes,is initiated by differentiation of these pluripotent cells to give riseto a population of mesenchymal precursor cells or mesenchymal stem cells(MSCs), which have the potential to differentiate into a variety ofmesodermal cell lineages such as for example, myoblasts, chondroblasts,osteoblasts and adipocytes. In the presence of appropriate environmentaland gene expression signals, the MSCs go through growth arrest anddifferentiate into precursors with a determined fate that undergo clonalexpansion, become committed and terminally differentiate to give rise tomature cells. The population of MSCs and more committed adiposeprogenitors that are found along with the stroma of adipose tissuecollectively are termed adipose-derived stem cells (ASCs). These cellshave a characteristic CD45⁻CD31⁻ CD34⁺CD105⁺ surface phenotype. In thecase of adipocyte differentiation, ASCs differentiate to proadipocytesthat undergo final differentiation to give rise to mature adipocytes.Mesenchymal progenitor cells with chondrogenic potential have also beenidentified in the infrapatellar fat pad in joints. (Lee et al., TissueEngg. 2010, 16(1): 317-325).

Table 3 lists cell lineages and respective inductive factors that can bederived from ASC lines. (Brown et. al., 2010, Plast. Reconstr. Surg.,126(6): 1936-1946; Gregoire et al., 1998, Physiol. Rev. 78(3): 783-809).

TABLE 3 Inductive Factors and Cell Lineages from Adipose-derived StemCells Cell Lineage Inductive Factors Adipocyte Dexamethasone; isobutylmethylxanthine,; indoxanthine; insulin; thiazolidinedione; nuclearhormone glucocorticoids, e.g., 3,3′,5-triiodothyronine (T₃) and retinoicacid (RA); IGF-1; PGE₂ Cardiomyocyte Transferrin; IL-3; IL-6; VEGFChondrocyte Ascorbic acid; bone morphogenetic protein 6; dexamethasone;insulin; transforming growth factor-β (TGF-β) Endothelial EGM-2-MVmedium (Cambrex, Walkersville, Md) containing ascorbate, epidermalgrowth factor, basic fibroblast growth factor, and hydrocortisoneMyocyte Dexamethasone horse serum Neuronal-like Butylatedhydroxianisole; valproic acid; insulin Osteoblast Ascorbic acid; bonemorphogenetic protein-2; dexamethasone; 1,25-dihydroxyvitamin D

Adipose Secreted Factors

Adipose is considered a secretory organ. The adipose secretome not onlyincludes structural and soluble factors contributing to the formation ofthe adipose matrix, but also a horde of soluble factors with endocrinefunction, such as growth factors, hormones, chemokines and lipids,collectively termed adipokines Exemplary adipokines include, withoutlimitation, leptin, adiponectin, resistin, interleukin 6 (IL-6),monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha(TNF-α); fibroblast growth factor (FGF), and vascular endothelial growthfactor (VEGF). Exemplary immunogical adipokines, particularly involvedin inflammatory pathways include, without limitation, serum amyloid A3(SAA3), IL-6, adiponectin, TNF-α and haptoglobin. Exemplary adipokinesinvolved in the production of new blood vessels include, withoutlimitation, angiopoietin-1, angiopoietin-2, VEGF, transforming growthfactor beta (TGF-β), hepatic growth factor (HGF), stromal derived growthfactor 1 (SDF-1), TNF-α, resistin, leptin, tissue factor, placentalgrowth factor (PGF), insulin like growth factor (IGF), and monobutyrin.

Adiponectin, a key metabolic factor secreted from adipocytes, is a30-KDa protein that may exist as a trimer, low molecular weight hexamersor high molecular weight 18mers. Adiponectin circulates throughout theplasma and has a variety of metabolic effects including, but not limitedto, glucose lowering and cardioprotection stimulation of smooth muscleproliferation. Adiponectin has been implicated in a number ofpathological conditions including, but not limited to diabetes, obesity,metabolic syndrome, cardiovascular disease and wound healing.

Resistin, a member of the resistin-like (RELM) hormone family, issecreted by stromal vascular cells of adipose. Resistin is secreted intwo multimeric isoforms and functions to counterbalance the insulinsensitizing effects of adiponectin. (Truillo, M. E. and Scherer P. E.,Endocrine Rev. 2006, 27(7): 762-778).

Secretions from resident adipocytes, macrophages and ASCs collectivelycontribute to the adipose secretome. Table 4 provides a reportedadipokine profile of ASCs. (Kilroy et. al., 2007, J. Cell. Physiol. 212:702-709.)

TABLE 4 Reported Adipokine Profile of Human ASCs Function AdipokineAngiogenic HGF VEGF Hematopoietic Flt-3 ligand G-CSF GM-CSF IL-7 IL-12M-CSF SCF Proinflammatory IL-1alpha IL-6 IL-8 IL-11 LIF TNF-alphaTranscription Factors Responsible for Adipogenesis

Adipocyte differentiation involves the crosstalk between externalsignals in the ECM environment with internal signals generated from thenucleus. The peroxisome proliferator-activated receptors (PPAR) andCCAAT-enhancer-binding proteins (C/EBP) family of transcription factorsplay an important role in adipogenesis. The PPARs, members of type IInuclear hormone receptor family, form heterodimers with the retinoid Xreceptor (RXR). They regulate transcription by binding of PPAR-RXRheteridimers to a response element characterized by a direct repeat ofthe nuclear receptor hexameric DNA recognition motif, PuGG-TCA. PPAR-γis most adipose-specific of all PPARs and is activated prior totranscriptional up-regulation of most other adipocyte genes. The C/EBPfamily of transcription factors are also induced prior to activation ofother adipocyte genes and plays a major role in adipocytedifferentiation. Members of the basic helix-loop-helix (bHLH) family oftranscription factors have also been implicated in adipogenesis.(Gregoire et al., 1998, Physiol. Rev. 78(3): 783-809).

2.2. Bone (Osseous) Tissue Compartment

Osseous tissue is a rigid form of connective tissue normally organizedinto definite structures, the bones. These form the skeleton, serve forthe attachment and protection of the soft parts, and, by theirattachment to the muscles, act as levers that bring about body motion.Bone is also a storage place for calcium that can be withdrawn whenneeded to maintain a normal level of calcium in the blood.

Bones can be classified according to their shape. Examples of bone typesinclude: long bones whose length is greater than their widths (e.g.,femur (thigh bone), humerus (long bone of the upper limb), tibia (shinbone), fibula (calf bone), radius (the outer of the two bones of theforearm), and ulna (inner of two bones of the forearm)), short boneswhose length and width is approximately equal (e.g., carpals bones(wrist bones in the hand)), flat bones (e.g., cranium (skull bonessurrounding the brain), scapula (shoulder blade), and ilia (theuppermost and largest bone of the pelvis)), irregular bones (e.g.,vertebra), and Sesamoid bones, small bones present in the joints toprotect tendons (fibrous connective tissues that connect muscles to thebones, e.g., patella bones (knee cap)).

Grossly, two types of bone may be distinguished: cancellous, trabecularor spongy bone, and cortical, compact, or dense bone.

Cortical bone, also referred to as compact bone or dense bone, is thetissue of the hard outer layer of bones, so-called due to its minimalgaps and spaces. This tissue gives bones their smooth, white, and solidappearance. Cortical bone consists of haversian sites (the canalsthrough which blood vessels and connective tissue pass in bone) andosteons (the basic units of structure of cortical bone comprising ahaversian canal and its concentrically arranged lamellae), so that incortical bone, bone surrounds the blood supply. Cortical bone has aporosity of about 5% to about 30%, and accounts for about 80% of thetotal bone mass of an adult skeleton.

Cancellous Bone (Trabecular or Spongy Bone)

Cancellous bone tissue, an open, cell-porous network also calledtrabecular or spongy bone, fills the interior of bone and is composed ofa network of rod- and plate-like elements that make the overallstructure lighter and allows room for blood vessels and marrow so thatthe blood supply surrounds bone. Cancellous bone accounts for theremaining 20% of total bone mass but has nearly ten times the surfacearea of cortical bone. It does not contain haversian sites and osteonsand has a porosity of about 30% to about 90%.

The head of a bone, termed the epiphysis, has a spongy appearance andconsists of slender irregular bone trabeculae, or bars, which anastomoseto form a lattice work, the interstices of which contain the marrow,while the thin outer shell appears dense. The irregular marrow spaces ofthe epiphysis become continuous with the central medullary cavity of thebone shaft, termed the diaphysis, whose wall is formed by a thin plateof cortical bone.

Both cancellous and cortical bone have the same types of cells andintercellular substance, but they differ from each other in thearrangement of their components and in the ratio of marrow space to bonesubstance. In cancellous bone, the marrow spaces are relatively largeand irregularly arranged, and the bone substance is in the form ofslender anastomosing trabeculae and pointed spicules. In cortical bone,the spaces or channels are narrow and the bone substance is denselypacked.

With very few exceptions, the cortical and cancellous forms are bothpresent in every bone, but the amount and distribution of each type varyconsiderably. The diaphyses of the long bones consist mainly of corticaltissue; only the innermost layer immediately surrounding the medullarycavity is cancellous bone. The tabular bones of the head are composed oftwo plates of cortical bone enclosing marrow space bridged by irregularbars of cancellous bone. The epiphyses of the long bones and most of theshort bones consist of cancellous bone covered by a thin outer shell ofcortical bone.

Each bone, except at its articular end, is surrounded by a vascularfibroelastic coat, the periosteum. The so-called endosteum, or innerperiosteum of the marrow cavity and marrow spaces, is not awell-demarcated layer; it consists of a variable concentration ofmedullary reticular connective tissue that contains osteogenic cellsthat are in immediate contact with the bone tissue.

Components of Bone

Bone is composed of cells and an intercellular matrix of organic andinorganic substances.

The organic fraction consists of collagen, glycosaminoglycans,proteoglycans, and glycoproteins. The protein matrix of bone largely iscomposed of collagen, a family of fibrous proteins that have the abilityto form insoluble and rigid fibers. The main collagen in bone is type Icollagen.

The inorganic component of bone, which is responsible for its rigidityand may constitute up to two-thirds of its fat-free dry weight, iscomposed chiefly of calcium phosphate and calcium carbonate, in the formof calcium hydroxyapatite, with small amounts of magnesium hydroxide,fluoride, and sulfate. The composition varies with age and with a numberof dietary factors. The bone minerals form long fine crystals that addstrength and rigidity to the collagen fibers; the process by which it islaid down is termed mineralization.

Bone Cells

Four cell types in bone are involved in its formation and maintenance.These are 1) osteoprogenitor cells, 2) osteoblasts, 3) osteocytes, and4) osteoclasts.

Osteoprogenitor Cells

Osteoprogenitor cells arise from mesenchymal cells, and occur in theinner portion of the periosteum and in the endosteum of mature bone.They are found in regions of the embryonic mesenchymal compartment wherebone formation is beginning and in areas near the surfaces of growingbones. Structurally, osteoprogenitor cells differ from the mesenchymalcells from which they have arisen. They are irregularly shaped andelongated cells having pale-staining cytoplasm and pale-staining nuclei.Osteoprogenitor cells, which multiply by mitosis, are identified chieflyby their location and by their association with osteoblasts. Someosteoprogenitor cells differentiate into osteocytes. While osteoblastsand osteocytes are no longer mitotic, it has been shown that apopulation of osteoprogenitor cells persists throughout life.

Osteoblasts

Osteoblasts, which are located on the surface of osteoid seams (thenarrow region on the surface of a bone of newly formed organic matrixnot yet mineralized), are derived from osteoprogenitor cells. They areimmature, mononucleate, bone-forming cells that synthesize collagen andcontrol mineralization. Osteoblasts can be distinguished fromosteoprogenitor cells morphologically; generally they are larger thanosteoprogenitor cells, and have a more rounded nucleus, a more prominentnucleolus, and cytoplasm that is much more basophilic. Osteoblasts makea protein mixture known as osteoid, primarily composed of type Icollagen, which mineralizes to become bone. Osteoblasts also manufacturehormones, such as prostaglandins, alkaline phosphatase, an enzyme thathas a role in the mineralization of bone, and matrix proteins.

Osteocytes

Osteocytes, star-shaped mature bone cells derived from ostoblasts andthe most abundant cell found in compact bone, maintain the structure ofbone. Osteocytes, like osteoblasts, are not capable of mitotic division.They are actively involved in the routine turnover of bony matrix andreside in small spaces, cavities, gaps or depressions in the bone matrixcalled lacuna. Osteocytes maintain the bone matrix, regulate calciumhomeostasis, and are thought to be part of the cellular feedbackmechanism that directs bone to form in places where it is most needed.Bone adapts to applied forces by growing stronger in order to withstandthem; osteocytes may detect mechanical deformation and mediatebone-formation by osteoblasts.

Osteoclasts

Osteoclasts, which are derived from a monocyte stem cell lineage andpossess phagocytic-like mechanisms similar to macrophages, often arefound in depressions in the bone referred to as Howship's lacunae. Theyare large multinucleated cells specialized in bone resorption. Duringresorption, osteoclasts seal off an area of bone surface; then, whenactivated, they pump out hydrogen ions to produce a very acidenvironment, which dissolves the hydroxyapatite component. The numberand activity of osteoclasts increase when calcium resorption isstimulated by injection of parathyroid hormone (PTH), while osteoclasticactivity is suppressed by injection of calcitonin, a hormone produced bythyroid parafollicular cells.

Bone Matrix

The bone matrix accounts for about 90% of the total weight of compactbone and is composed of microcrystalline calcium phosphate resemblinghydroxyapatite (60%) and fibrillar type I collagen (27%). The remaining3% consists of minor collagen types and other proteins includingosteocalcin, osteonectin, osteopontin, bone sialoprotein, as well asproteoglycans, glycosaminoglycans, and lipids.

Bone matrix is also a major source of biological information thatskeletal cells can receive and act upon. For example, extracellularmatrix glycoproteins and proteoglycans in bone bind a variety of growthfactors and cytokines, and serve as a repository of stored signals thatact on osteoblasts and osteoclasts. Examples of growth factors andcytokines found in bone matrix include, but are not limited to, BoneMorphogenic Proteins (BMPs), Epidermal Growth Factors (EGFs), FibroblastGrowth Factors (FGFs), Platelet-Derived Growth Factors (PDGFs),Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factors(TGFs), Bone-Derived Growth Factors (BDGFs), Cartilage-Derived GrowthFactor (CDGF), Skeletal Growth Factor (hSGF), Interleukin-1 (IL-1), andmacrophage-derived factors.

There is an emerging understanding that extracellular matrix moleculesthemselves can serve regulatory roles, providing both direct biologicaleffects on cells as well as key spatial and contextual information.

The Periosteum and Endosteum

The periosteum is a fibrous connective tissue investment of bone, exceptat the bone's articular surface. Its adherence to the bone varies bylocation and age. In young bone, the periosteum is stripped off easily.In adult bone, it is more firmly adherent, especially so at theinsertion of tendons and ligaments, where more periosteal fiberspenetrate into the bone as the perforating fibers of Sharpey (bundles ofcollagenous fibers that pass into the outer circumferential lamellae ofbone). The periosteum consists of two layers, the outer of which iscomposed of coarse, fibrous connective tissue containing few cells butnumerous blood vessels and nerves. The inner layer, which is lessvascular but more cellular, contains many elastic fibers. During growth,an osteogenic layer of primitive connective tissue forms the inner layerof the periosteum. In the adult, this is represented only by a row ofscattered, flattened cells closely applied to the bone. The periosteumserves as a supporting bed for the blood vessels and nerves going to thebone and for the anchorage of tendons and ligaments. The osteogeniclayer, which is considered a part of the periosteum, is known to furnishosteoblasts for growth and repair, and acts as an important limitinglayer controlling and restricting the extend of bone formation. Becauseboth the periosteum and its contained bone are regions of the connectivetissue compartment, they are not separated from each other or from otherconnective tissues by basal laminar material or basement membranes.Perosteal stem cells have been shown to be important in boneregeneration and repair. (Zhang et al., 2005, J. Musculoskelet.Neuronal. Interact. 5(4): 360-362).

The endosteum lines the surface of cavities within a bone (marrow cavityand central canals) and also the surface of trabeculae in the marrowcavity. In growing bone, it consists of a delicate striatum ofmyelogenous reticular connective tissue, beneath which is a layer ofosteoblasts. In the adult, the osteogenic cells become flattened and areindistinguishable as a separate layer. They are capable of transforminginto osteogenic cells when there is a stimulus to bone formation, asafter a fracture.

Marrow

The marrow is a soft connective tissue that occupies the medullarycavity of the long bones, the larger central canals, and all of thespaces between the trabeculae of spongy bone. It consists of a delicatereticular connective tissue, in the meshes of which lie various kinds ofcells. Two varieties of marrow are recognized: red and yellow. Redmarrow is the only type found in fetal and young bones, but in the adultit is restricted to the vertebrae, sternum, ribs, cranial bones, andepiphyses of long bones. It is the chief site for the genesis of bloodcells in the adult body. Yellow marrow consists primarily of fat cellsthat gradually have replaced the other marrow elements. Under certainconditions, the yellow marrow of old or emaciated persons loses most ofits fat and assumes a reddish color and gelatinous consistency, known asgelatinous marrow. With adequate stimulus, yellow marrow may resume thecharacter of red marrow and play an active part in the process of blooddevelopment.

Osteogenesis or Ossification

Osteogenesis or ossification is a process by which the bones are formed.There are three distinct lineages that generate the skeleton. Thesomites generate the axial skeleton, the lateral plate mesodermgenerates the limb skeleton, and the cranial neural crest gives rise tothe branchial arch, craniofacial bones, and cartilage. There are twomajor modes of bone formation, or osteogenesis, and both involve thetransformation of a preexisting mesenchymal tissue into bone tissue. Thedirect conversion of mesenchymal tissue into bone is calledintramembranous ossification. This process occurs primarily in the bonesof the skull. In other cases, mesenchymal cells differentiate intocartilage, which is later replaced by bone. The process by which acartilage intermediate is formed and replaced by bone cells is calledendochondral ossification.

Intramembranous Ossification

Intramembraneous ossification is the characteristic way in which theflat bones of the scapula, the skull and the turtle shell are formed. Inintramembraneous ossification, bones develop sheets of fibrousconnective tissue. During intramembranous ossification in the skull,neural crest-derived mesenchymal cells proliferate and condense intocompact nodules. Some of these cells develop into capillaries; otherschange their shape to become osteoblasts, committed bone precursorcells. The osteoblasts secrete a collagen-proteoglycan matrix that isable to bind calcium salts. Through this binding, the prebone (osteoid)matrix becomes calcified. In most cases, osteoblasts are separated fromthe region of calcification by a layer of the osteoid matrix theysecrete. Occasionally, osteoblasts become trapped in the calcifiedmatrix and become osteocytes. As calcification proceeds, bony spiculesradiate out from the region where ossification began, the entire regionof calcified spicules becomes surrounded by compact mesenchymal cellsthat form the periosteum, and the cells on the inner surface of theperiosteum also become osteoblasts and deposit osteoid matrix parallelto that of the existing spicules. In this manner, many layers of boneare formed.

Intramembraneous ossification is characterized by invasion ofcapillaries into the mesenchymal zone, and the emergence anddifferentiation of mesenchymal cells into mature osteoblasts, whichconstitutively deposit bone matrix leading to the formation of bonespicules, which grow and develop, eventually fusing with other spiculesto form trabeculae. As the trabeculae increase in size and number theybecome interconnected forming woven bone (a disorganized weak structurewith a high proportion of osteocytes), which eventually is replaced bymore organized, stronger, lamellar bone.

The molecular mechanism of intramembranoaus ossification involves bonemorphogenetic proteins (BMPs) and the activation of a transcriptionfactor called CBFA1. Bone morphogenetic proteins, for example, BMP2,BMP4, and BMP7, from the head epidermis are thought to instruct theneural crest-derived mesenchymal cells to become bone cells directly.BMPs activate the Cbfa1 gene in mesenchymal cells. The CBFA1transcription factor is known to transform mesenchymal cells intoosteoblasts. Studies have shown that the mRNA for mouse CBFA1 is largelyrestricted to the mesenchymal condensations that form bone, and islimited to the osteoblast lineage. CBFA1 is known to activate the genesfor osteocalcin, osteopontin, and other bone-specific extracellularmatrix proteins.

Endochondral Ossification (Intracartilaginous Ossification)

Endochondral ossification, which involves the in vivo formation ofcartilage tissue from aggregated mesenchymal cells, and the subsequentreplacement of cartilage tissue by bone, can be divided into fivestages. The skeletal components of the vertebral column, the pelvis, andthe limbs are first formed of cartilage and later become bone.

First, the mesenchymal cells are committed to become cartilage cells.This commitment is caused by paracrine factors that induce the nearbymesodermal cells to express two transcription factors, Pax1 andScleraxis. These transcription factors are known to activatecartilage-specific genes. For example, Scleraxis is expressed in themesenchyme from the sclerotome, in the facial mesenchyme that formscartilaginous precursors to bone, and in the limb mesenchyme.

During the second phase of endochondral ossification, the committedmesenchyme cells condense into compact nodules and differentiate intochondrocytes (cartilage cells that produce and maintain thecartilaginous matrix, which consists mainly of collagen andproteoglycans). Studies have shown that N-cadherin is important in theinitiation of these condensations, and N-CAM is important formaintaining them. In humans, the SOX9 gene, which encodes a DNA-bindingprotein, is expressed in the precartilaginous condensations.

During the third phase of endochondral ossification, the chondrocytesproliferate rapidly to form the model for bone. As they divide, thechondrocytes secrete a cartilage-specific extracellular matrix.

In the fourth phase, the chondrocytes stop dividing and increase theirvolume dramatically, becoming hypertrophic chondrocytes. These largechondrocytes alter the matrix they produce (by adding collagen X andmore fibronectin) to enable it to become mineralized by calciumcarbonate.

The fifth phase involves the invasion of the cartilage model by bloodvessels. The hypertrophic chondrocytes die by apoptosis, and this spacebecomes bone marrow. As the cartilage cells die, a group of cells thathave surrounded the cartilage model differentiate into osteoblasts,which begin forming bone matrix on the partially degraded cartilage.Eventually, all the cartilage is replaced by bone. Thus, the cartilagetissue serves as a model for the bone that follows.

The replacement of chondrocytes by bone cells is dependent on themineralization of the extracellular matrix. A number of events lead tothe hypertrophy and mineralization of the chondrocytes, including aninitial switch from aerobic to anaerobic respiration, which alters theircell metabolism and mitochondrial energy potential. Hypertrophicchondrocytes secrete numerous small membrane-bound vesicles into theextracellular matrix. These vesicles contain enzymes that are active inthe generation of calcium and phosphate ions and initiate themineralization process within the cartilaginous matrix. The hypertrophicchondrocytes, their metabolism and mitochondrial membranes altered, thendie by apoptosis.

In the long bones of many mammals (including humans), endochondralossification spreads outward in both directions from the center of thebone. As the ossification front nears the ends of the cartilage model,the chondrocytes near the ossification front proliferate prior toundergoing hypertrophy, pushing out the cartilaginous ends of the bone.The cartilaginous areas at the ends of the long bones are calledepiphyseal growth plates. These plates contain three regions: a regionof chondrocyte proliferation, a region of mature chondrocytes, and aregion of hypertrophic chondrocytes. As the inner cartilagehypertrophies and the ossification front extends farther outward, theremaining cartilage in the epiphyseal growth plate proliferates. As longas the epiphyseal growth plates are able to produce chondrocytes, thebone continues to grow.

Bone Remodeling

Bone constantly is broken down by osteoclasts and re-formed byosteoblasts in the adult. It has been reported that as much as 18% ofbone is recycled each year through the process of renewal, known as boneremodeling, which maintains bone's rigidity. The balance in this dynamicprocess shifts as people grow older: in youth, it favors the formationof bone, but in old age, it favors resorption.

As new bone material is added peripherally from the internal surface ofthe periosteum, there is a hollowing out of the internal region to formthe bone marrow cavity. This destruction of bone tissue is due toosteoclasts that enter the bone through the blood vessels. Osteoclastsdissolve both the inorganic and the protein portions of the bone matrix.Each osteoclast extends numerous cellular processes into the matrix andpumps out hydrogen ions onto the surrounding material, therebyacidifying and solubilizing it. The blood vessels also import theblood-forming cells that will reside in the marrow for the duration ofthe organism's life.

The number and activity of osteoclasts must be tightly regulated. Ifthere are too many active osteoclasts, too much bone will be dissolved,and osteoporosis will result. Conversely, if not enough osteoclasts areproduced, the bones are not hollowed out for the marrow, andosteopetrosis (known as stone bone disease, a disorder whereby the bonesharden and become denser) will result.

Bone Regeneration and Fracture Repair

A fracture, like any traumatic injury, causes hemorrhage and tissuedestruction. The first reparative changes thus are characteristic ofthose occurring in any injury of soft tissue. Proliferating fibroblastsand capillary sprouts grow into the blood clot and injured area, thusforming granulation tissue. The area also is invaded by polymorphonuclear leukocytes and later by macrophages that phagocytize thetissue debris. The granulation tissue gradually becomes denser, and inparts of it, cartilage is formed. This newly formed connective tissueand cartilage is designated as a callus. It serves temporarily instabilizing and binding together the fracture bone. As this process istaking place, the dormant osteogenic cells of the periosteum enlarge andbecome active osteoblasts. On the outside of the fractured bone, atfirst at some distance from the fracture, osseous tissue is deposited.This formation of new bone continues toward the fractured ends of thebone and finally forms a sheath-like layer of bone over thefibrocartilaginous callus. As the amount of bone increases, osteogenicbuds invade the fibrous and cartilaginous callus and replace it with abony one. The cartilage undergoes calcification and absorption in thereplacement of the fibrocartilaginous callus and intramembraneous boneformation also takes place. The newly formed bone is at first a spongyand not a compact type, and the callus becomes reduced in diameter. Atthe time when this subperiosteal bone formation is taking place, bonealso forms in the marrow cavity. The medullary bone growingcentripetally from each side of the fracture unites, thus aiding thebony union.

The process of repair is, in general, an orderly process, but it variesgreatly with the displacement of the fractured ends of the bone and thedegree of trauma inflicted. Uneven or protruding surfaces gradually areremoved, and the healed bone, especially, in young individuals, assumesits original contour.

Osteogenesis and Angiogenesis

Skeletal development and fracture repair includes the coordination ofmultiple events such as migration, differentiation, and activation ofmultiple cell types and tissues. The development of a microvasculatureand microcirculation is important for the homeostasis and regenerationof living bone, without which the tissue would degenerate and die.Recent developments using in vitro and in vivo models of osteogenesisand fracture repair have provided a better understanding of therecruitment nature of the vasculature in skeletal development andrepair.

The vasculature transports oxygen, nutrients, soluble factors andnumerous cell types to all tissues in the body. The growth anddevelopment of a mature vascular structure is one of the earliest eventsin organogenesis. In mammalian embryonic development, the nascentvascular networks develop by aggregation of de novo forming angioblastsinto a primitive vascular plexus (vasculogenesis). This undergoes acomplex remodeling process in which sprouting, bridging and growth fromexisting vessels (angiogenesis) leads to the onset of a functionalcirculatory system.

The factors and events that lead to the normal development of theembryonic vasculature are recapitulated during situations ofneoangiogenesis in the adult. There are a number of factors involved inneoangiogenesis; these include, but are not limited to, VascularEndothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF),various members of the Transforming Growth factor beta (TGFβ) family andHypoxia-Inducible Transcription Factor (HIF). Other factors that haveangiogenic properties include the Angiopoietins, (Ang-1); HepatocyteGrowth Factor (HGF); Platelet-Derived Growth Factor (PDGF); Insulin-likeGrowth Factor family (IGF-1, IGF-2) and the Neurotrophins (NGF).

The VEGFs and their corresponding receptors are key regulators in acascade of molecular and cellular events that ultimately lead to thedevelopment of the vascular system, either by vasculogenesis,angiogenesis or in the formation of the lymphatic vascular system.Although VEGF is a critical regulator in physiological angiogenesis, italso plays a significant role in skeletal growth and repair.

In the mature established vasculature, the endothelium plays animportant role in the maintenance of homeostasis of the surroundingtissue by providing the communicative network to neighboring tissues torespond to requirements as needed. Furthermore, the vasculature providesgrowth factors, hormones, cytokines, chemokines and metabolites, and thelike, needed by the surrounding tissue and acts as a barrier to limitthe movement of molecules and cells. Signals and attractant factorsexpressed on the bone endothelium help recruit circulating cells,particularly hematopoietic cells, to the bone marrow and coordinate withmetastatic cells to target them to skeletal regions. Thus, anyalteration in the vascular supply to bone tissue can lead to skeletalpathologies, such as osteonecrosis (bone death caused by reduced bloodflow to bones), osteomyelitis (infection of the bone or bone marrow bymicroorganism), and osteoporosis (loss of bone density). A number offactors have been found to have a prominent effect on the pathology ofthe vasculature and skeleton, including Osteoprotegerin (OPG), whichinhibits Receptor Activator of NF-κB Ligand (RANKL)-inducedosteoclastogenic bone resorption.

Both intramembraneous and endochondral bone ossification occur in closeproximity to vascular ingrowth. In endochondral ossification, thecoupling of chondrogenesis and osteogenesis to determine the rate ofbone ossification is dependent on the level of vascularization of thegrowth plate. For example, vascular endothelial growth (VEGF) factorisoforms are essential in coordinating metaphyseal and epiphysealvascularization, cartilage formation, and ossification duringendochondral bone development. HIF-1 stimulates transcription of theVEGF gene (and of other genes whose products are needed when oxygen isin short supply). The VEGF protein is secreted, diffuses through thetissue, and acts on nearby endothelial cells.

The response of the endothelial cells includes at least four components.First, the cells produce proteases to digest their way through the basallamina of the parent capillary or venule. Second, the endothelial cellsmigrate toward the source of the signal. Third, the cells proliferate.Fourth, the cells form tubes and differentiate. VEGF acts on endothelialcells selectively to stimulate this entire set of effects. Other growthfactors, including some members of the fibroblast growth factor family,also can stimulate angiogenesis, but they influence other cell typesbesides endothelial cells. As the new vessels form, bringing blood tothe tissue, the oxygen concentration rises, HIF-1 activity declines,VEGF production is shut off, and angiogenesis ceases.

The vascularization of cartilage regions in long bones occurs atdifferent stages of development. In early embryonic development, bloodvessels that originate from the perichondrium invaginate into thecartilage structures. During elevated postnatal growth, capillariesinvade the growth plate of long bones. In adulthood, angiogenesisperiodically can be switched on during bone remodeling in response tobone trauma or pathophysiological conditions such as rheumatoidarthritis (RA) and osteoarthritis (OA).

Bone has the unique capacity to regenerate without the development of afibrous scar, which is symptomatic of soft tissue healing of wounds.This is achieved through the complex interdependent stages of thehealing process, which mimic the tightly regulated development of theskeleton. Following trauma with damage to the musculoskeletal system,disruption of the vasculature leads to acute necrosis and hypoxia of thesurrounding tissue. This disruption of the circulation leads to theactivation of thrombotic factors in a coagulation cascade leading to theformation of a hematoma. The inflammatory response and tissue breakdownactivate factors such as cytokines and growth factors that recruitosteoprogenitor and mesenchymal cells to the fracture site. Thestimulation of the endosteal circulation in the fractured bone allowsmesenchymal cells associated with growing capillaries to invade thewound region from the endosteum and bone marrow. At the edge of a bonefracture, the transiently formed granulation tissue is replaced byfibrocartilage. Concomitantly, the periosteum directly undergoesintramembranous bone formation leading to the formation of an externalcallus; while internally, the tissue is being mineralized to form wovenbone. After stabilization of the bone tissue and vasculature in the bonefracture, the cell mediated remodeling cascade is activated whereosteoclastic removal of necrotic bone is followed by the replacement ofthe large fracture callus by lamellar bone, the callus size is reducedand the normal vascular supply is restored.

A plurality of mediators associated with fetal and postnatal bonedevelopment plays a prominent role in the cascade response in bonefracture repair. These include but are not limited to BMP-2 and 4, VEGF,bFGF, TGF-β, and PDGF. VEGF expression is detected on chondroblasts,chondrocytes, osteoprogenitor cells and osteoblasts in the fracturecallus where it is highly expressed in angioblasts, osteoprogenitor andosteoblast cells during the first seven days of healing but decreasesafter eleven days. Additionally, osteoclasts release heparinase thatinduces the release of the active form of VEGF from heparin, activatingnot only angiogenesis but also osteoclast recruitment, differentiationand activity leading to the remodeling of the fracture callus duringendochondral ossification. Fractures in some cases fail to repair orunite resulting in fibrous filled pseudarthrosis. A number ofcontributing factors can lead to non-union or delayed union of bonefractures, such as, but not limited to, anti-inflammatory drugs,steroids, Vitamin C, Vitamin D and calcium deficiencies, tobaccosmoking, diabetes, and other physiological disorders.

The absence of a functional vascular network is also an important factorin the lack of bone healing in non-union fractures. Studies havereported that angiogenic factors released from biomimetic scaffolds canenhance bone regeneration and that combination strategies that releaseboth angiogenic and osteogenic factors can enhance the regenerativecapacity of bone.

The critical sequential timing of osteoclast differentiation andactivation, angiogenesis, recruitment of osteoprogenitor cells and therelease of growth factors such as BMP-2 in osteogenesis and fracturerepair may be enhanced by the synchronized endogenous production ofangiogenic and osteogenic mediators. Studies in rat femoral drill-holeinjury have shown differential expression of VEGF splicing isoformsalong with its receptors, indicating an important role in the bonehealing process. Other studies have demonstrated that angiogenesisoccurs predominantly before the onset of osteogenesis in bonelengthening in an osteodistraction model.

Another angiogenic inducing growth factor, FGF-2, can acceleratefracture repair when added exogenously to the early healing stage of abone. Although the mechanism has not been fully elucidated, it has theability to stimulate angiogenesis and the proliferation anddifferentiation of osteoblasts to possibly aid the repair of bonefractures.

2.3. Cartilaginous Tissue Compartments

Cartilaginous tissue compartments are specialized connective tissuecompartments comprising cartilage cells, known as chondrocytes,cartilage fibers and ground substance constituting the cartilage matrix,that collectively contribute to characteristic elastic firmnessrendering cartilage capable of withstanding high levels of pressure orsheer. Cartilage is histologically classified into three types dependingon its molecular composition: hyaline cartilage; fibrocartilage andelastic cartilage.

Hyaline cartilage is the predominant form of cartilage comprising anamorphous matrix surrounding chondrocytes embedded within spaces, knownas lacunae. Hyaline cartilage, which is commonly associated with theskeletal system and found in the nose, trachea, bronchi and larynx,predominantly functions to provide support. Hyaline cartilage associatedwith the articular portions of bone, forming the major component ofsynovial joints, is termed articular cartilage. Hyaline cartilage isusually avascular except where vessels may pass through to supply othertissues and in ossification centers involved in intracartilaginous bonedevelopment.

Fibrocartilage, which is commonly found in intervertebral discs andpubic symphysis and functions to provide tensile strength and in shockabsorption, is less firm than hyaline cartilage. It comprises acombination of dense collagenous fibers with cartilage cells and a scantcartilage matrix. Fibrocartilage is not usually circumscribed by aperichondrium. Proportions of cells, fibers and ECM components infribrocartilage are variable.

Elastic cartilage, which is found in the external ear, the Eustachiantube, epiglottis and some of the lanryngeal cartilages, is characterizedby a large number of elastic fibers that branch and course in alldirections to form a dense network of anastomising and interlacingfibers.

Articular Cartilage Matrix

The chondrocytes in articular cartilage are surrounded by a narrowregion of connective tissue ECM, termed the pericellular matrix (PCM),which together with the chondrocyte, is termed chondron. The PCM, whichis very rich in fibronectin, proteoglycans (e.g., aggrecan, hyaluron anddecorin) and collagen (types II, VI and IX), is particularlycharacterized by a high concentration of type VI collagen as compared tothe surrounding ECM. In normal articular cartilage, type VI collagen isrestricted to the chondrons, but in osteoarthritic cartilage, it isupregulated and found throughout the ECM. A proteomic analysis ofarticular cartilage revealed the presence of collagen α1(II)C-propeptide, collagen α1(XI) C-propeptide, collagen α2(XI)C-propeptide, collagen α1(VI), collagen α2(VI), link protein, biglycan,decorin, osteonectin, matrillin-1, annexin-V, lactadherin, and bindingimmunoglobulin protein (BiP), in addition to metabolic proteins. (Wilsonet. al., 2008, Methods, 48: 22-31).

Chondrocyte Differentiation

The specific structure of articular cartilage, with endogenouschondrocytes forming adult joints, is the result of endochondralossification, as described above under the Heading, Osseous TissueCompartments Formation.

Chondrocyte differentiation and maintenance in articular cartilage isgoverned by interaction of multiple factors. Key players include, butare not limited to, ions (e.g., calcium); steroids (e.g., estrogens);terpenoids (e.g., retinoic acid); peptides (e.g., Parathyroid hormone(PTH), parathyroid hormone-related peptide (PTHrP)), insulin growthfactors (e.g., TGFβ hormones, including, without limitation, BMPs,IGF-1, VEGF, PDGF, FGF); transcription factors (e.g., Wnt, SOX-9);eicosanoids (e.g., prostaglandins); catabolic interleukins (e.g., IL-1);and anabolic interleukins (e.g., IL-6, IL-4 and IL-10). (Gaissmaier etal., 2008, Int. J. Care Injured, 39S1: S88-S96).

Growth Plate

The epiphyseal plates or growth plates are a hyaline cartilage platelocated in the metaphysis at the end of long bones. Whereas endochondralossification is responsible for the formation of cartilage in utero andin infants, the growth plates are responsible for the longitudinalgrowth of long bones via a cartilage template. The ongoing developmentalprocesses of proliferation and differentiation within the growth platesare mediated by a number of hormonal and paracrine factors secreted bythe growth plate chondrocytes. The growth plate is a highly organizedstructure comprising a large number of chondrocytes in various stages ofdifferentiation and proliferation embedded in a scaffold of ECMcomponents.

The growth plate can be subdivided into four zones depending on thestage of differentiation and spatial distribution of collagen types. Theresting zone is the smallest zone close to the epiphyseal cartilagecomprising small monomorphic chondrocytes with a narrow rim ofcytoplasm. The chondrocytes of the resting zone secrete growth plateorienting factor (GPOF) that aligns proliferating cells parallel to thelong axis of the developing bone. Stem cell-like cells of the restingzone have a limited proliferative capacity, which eventually leads tofusion of the growth plate (epiphyseal fusion). The proliferative zoneof the growth plate comprises chondrocytes that are arranged incharacteristic columns parallel to the longitudinal axis of the bone andare separated by ECM with high type II collagen. The chondrocytes of theproliferative zone are mitotically active, have high oxygen and glycogencontent, and exhibit increased mitochondrial ATP production. Thehypertrophic zone refers to the zone farthest from the resting zonewhere prehypertrophic chondrocytes stop dividing and terminallydifferentiate into elongated hypertrophic chondrocytes embedded in ECMhigh in type X collagen. Hypertrophic chondrocytes have a highintracellular calcium concentration required for the production ofrelease vesicles containing Ca²⁺-binding annexins, that secrete calciumphosphate, hydroxyapatite, phosphatases (such as alkaline phosphatase),metalloproteinases, all instrumental in proteolytic remodeling andmineralization of the surrounding matrix. The hypertrophic chondrocytesproduce factors, such as VEGF, that initiate vascularization of themineralized matrix that is then degraded by invading phagocyticchondroclasts and osteoclasts constituting the invading zone.

The developmental processes involving chondrogenesis are regulated by aninterplay of a large number of systemic hormones and paracrine factors,including growth factors, cytokines and transcription factors. Table 5lists key factors involved in chondrocyte proliferation anddifferentiation in the growth plate. (Brochhausen et al., J. Tissue Eng.Regen. Med. 2009, 3: 416-429).

TABLE 5 Summary of Key Factors involved in Chondrocyte Proliferation andDifferentiation in the Growth Plate Name Class Expression Effect ATF-2Transcription factor Resting chondrocytes; Apoptosis proliferativechondrocytes Bcl-2 Inner mitochondrial Proliferative chondrocytes;Apoptosis membrane protein prehypertrophic chondrocytes Ihh Signalingmolecule Prehypertrophic chondrocytes Proliferation PTHrP Peptidehormone Perichondrium perarticular Proliferation chondrocytes BMP TGF-βsuperfamily Prehypertrophic chondrocytes Cartilage growth factorsformation; proliferation PGE₂ Lipid mediator All zones of growth plateProliferation matrix synthesis MMP Metalloproteinase Hypertrophicchondrocytes; Apoptosis; chondroclasts vascularization matrixdegradation Sox Transcription factor Resting and proliferativeDifferentiation; chondrocytes; hypertrophic proliferation; chondrocytesRunx 2 Transcription factor Hypertrophic chondrocytes Terminal (Cbfa 1)differentiation; matrix mineralization NOTCH Single pass Prehypertrophicand Inhibits terminal transmembrane protein hypertrophic chondrocytesdifferentiation HOX Homeobox transcription Hypertrophic chondrocytesActivates factors osteogenic genes FGF Fibroblast growth factorProliferative chondrocytes Antiproliferation

Stem Cells of Cartilaginous Tissue Compartments

Multipotent mesenchymal progenitor cells with adipogenic, osteogenic andchondrogenic potential, and that are CD105+/CD166+(corresponding toTGF-β type III receptor (endoglin) and ALCAM, respectively), have beenidentified in articular cartilage. (Asalameh et al., Arthritis &Rheumatism, 2004, 50(5): 1522-1532). The presence ofCD34−/CD45−/CD44+/CD73+/CD90+ mesenchymal stem cells with adipogenic,chondrogenic and osteogenic potential also has been shown. (Peng et al.,Stem Cells and Development (2008), 17: 761-774). Similar to bone-derivedMSCs, articular-derived MSCs are positive for surface expression ofNotch-1. (Hiraoka et al., Biorheology, 2006, 43: 447-454). A potentialMSC niche positive for Stro-1, Jagged-1 and BMPr1a has also beenidentified in the perichondrial zone of Ranvier on the growth plate.(Karlsson et al., 2009, J. Anat. 215(3): 355-63).

Differential expression of Notch-1, Stro-1 and VCAM-1/CD106 markers hasbeen observed in normal articular cartilage versus osteoarthritic (OA)cartilage. In normal cartilage, expression of these markers is higher inthe superficial zone (SZ) as compared to the middle zone (MZ) and deepzone (DZ). On the other hand, OA cartilage SZ has reduced Notch-1 andSox-9 while MZ has increased Notch-1, Stro-1 and VCAM-1 positive cells.(Grogan et al., Arthritis Res. Ther. 2009, 11(3): R85-R97).

Intervertebral Disc Fibrocartilage Tissue Compartments

The intervertebral discs (IVD) predominantly are comprised offibrocartilage. The IVD fibrocartilage is continuous both with and belowthe articular cartilage of adjacent vertebrae as well as peripherallywith spinal ligaments. The IVD is a unique structure containing annulusfibrosus (AF) and nucleus pulposus (NP), a gelatinous ellipsoidalremnant of the embryonic notochord, and is sandwiched between twoadjacent cartilaginous endplates (EP). IVD rupture and herniation of thenucleus pulposus into the spinal cord may cause severe pain and otherneurological symptoms. The NP and AF synergistically function to achievethe primary role of IVD in transferring load, dissipating energy andfacilitating in joint mobility.

The adult IVD is essentially avascular; hence, endogenous cells survivein a low-nutrient and low-oxygen microenvironment. The major ECMcomponents of IVD include but are not limited to aggrecan, collagen(e.g., types I, II and IX), leucine rich repeat (LRR) proteins andproteoglycans (e.g., fibromodulin, decorin, lumican), cartilageoligomatrix protein, and collagen VI beaded filament network. (Feng etal., 2006, J. Bone Joint Surg. Am. 88: 25-29). The water content, GAGcontent, aggrecan levels and levels of type II collagen aresignificantly lower in older discs demonstrating the effects of IVDdegeneration with age. (Murakami et al., 2010, Med. Biol. Eng. Comput.48: 469-474).

The central nucleus pulposus (NP) is rich in aggrecan and hyaluron. Thedeveloping NP is characterized by the presence of highly vacuolatedchondrocytes and small chondroblasts inherited from the notochord.Primarily functioning as a primitive axial support, the integrity of thenotochord is maintained by a proteoglycan (PG-) and laminin-rich sheath.As NP matures, the cellular composition becomes predominantlychondrocytic. Mature NP cells are small and have an aggrecan richmatrix, which is essential in maintaining requisite hydration levels formechanical function. Their gene expression profile and metabolicactivity are distinct from the chondrocytes of articular cartilage. TheECM of immature NP has high aggrecan levels and primarily contains typeII collagen, with the type IIA isoform expressed by progenitor cellsduring chondrogenesis, not by mature chondrocytes. (Hsieh A. H. andTworney J. D., J. Biomech., 2010, 43(1): 137-156).

The AF surrounds the NP with layers of unidirectional sheets of collagenparallel to the circumference of a disc to form collagen lamellae.Alternating bidirectional collagen fibers intersperse the AF collagenlamellae. AF can be subdivided into three regions: inner AF, middle AFand outer AF. The inner AF arises along with endochondral formation ofthe vertebrae. The outer AF arises as a separate cell condensation withslower matrix formation. Lamellae of inner AF comprises predominantly oftype II collagen and fibrochondrocytes, while those of outer AF arecomprised of type I collagen and fibroblasts. A population of pancakeshaped interlamellar cells as well as elastin fibers are also foundwithin the lamellae, in vertebral attachments, and at the NP-AFinterface. Large proteoglycans (PGs; for example aggrecan and versican)and type I and VI collagen permeate interlamellar and translamellar ECM.(Hsieh A. H. and Tworney J. D., J. Biomech., 2010, 43(1): 137-156).

A large number of coordinated signals originating from the cells of thenotochord and floor plate of the embryonal neural tube are instrumentalin disc embryogenesis. Key signals include, but are not limited to,sonic hedgehog (Shh), Wnt, noggin, Pax family of transcription factors(e.g., Pax 1 and Pax 9), Sox family of transcription factors (Sox5, Sox6and Sox) and TGF-β. (Smith et al., 2011, Dis Model Mech. 4(1): 31-41).Herniation and IVD degeneration are associated with changes ininflammatory and immune cytokine profiles, including, but not limitedto, the activation of Th1-related cytokines (e.g. IFNγ) as well asTh17-related cytokines (e.g., IL-4, IL-6, IL-12 and IL-17). (Shamji etal., 2010, Arthritis & Rheumatism, 62(7): 1974-1982).

A potential stem cell niche comprised of progenitor cells that arepositive for Notch1, Delta4, Jagged1, CD117, Stro-1 and Ki67 has beenidentified in intervertebral discs of a number of animals, includinghumans. It has been reported that the IVD tissue compartments comprise aslow growing zone in the AF as well as the NP regions. (Henriksson etal., 2009, SPINE, 34(21): 2278-2287).

2.4. Dental Tissue Compartments

A tooth has three anatomical divisions (crown, root and neck), and fourstructural components (enamel, dentin, cementum and pulp).

Enamel is the hardest, most mineralized biological tissue in the humanbody. It is composed of elongated hydroxyapatite crystallites bundledinto rods or prisms, interspersed with crystalline interrods filling theinterstitial space. Enamel cells, known as ameloblasts, are responsiblefor enamel development. Ameloblastin, TRAP and enamelin are key proteinsfound in enamel tissue whereas the enamel matrix is devoid of collagen,composed primarily of amelogenin. An intricate orchestration ofsignaling factors, such as BMPs (e.g., BMP-2, BMP-4, BMP-7), FGFs (e.g.,FGF-3, -4, -9, -20), Wnt-3, 10a, 10b and transcription factors, such as,p21, Msx2 and Lef1 is responsible for morphogenesis of enamel.Self-assembly of amelogens to form amelogenin nanospheres play a role innucleation of hydroxyapatite crystallization and enamel mineralization.Matrix processing enzymes, such as MMP-20, kallikrein-4 (KLK4), alsoknown as enamel matrix serine protease-1 (EMSP-1), are involved in thecomplete elimination of the protein matrix and replacement with amineralized matrix. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).Ameloblasts arise from epithelial stem cells of ectodermal origin. Theyare lost after tooth eruption leaving no adult human ectodermal stemcells in the mature enamel. In contrast, rodent enamel retain a niche ofepithelial stem cells, known as apical bud cells, for continuous enamelproduction. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed,120:860-872).

Dentin is a hard, yellowish and elastic living connective tissuecompartment with biomechanical properties similar to bone. The formationof dentin is driven by mesenchymally derived mature odontoblasts thatare fully differentiated and nondividing and that form a single layerunderneath the dentin in a mature tooth. A series ofepithelial-mesenchymal interactions regulates odontoblastdifferentiation from neural crest cells in the first branchial arch andfrontonasal processes. Mature dentin is comprised of a mantle, composedof intertubular and peritubular dentin made of a collagen fibril matrix,with odontoblast cell processes extending into dentin tubules. Duringdentinogenesis, odontoblasts secrete predentin, a mineralized tissuecomposed of type I collagen. Unlike osteogenesis, in dentinogenesis, asthe predentin layer is formed, the odontoblasts recede instead ofbecoming embedded within the dentin matrix, leaving behind cellsprocesses within dentinal tubules. Subsequently, the unmineralizedpredentin is converted to dentin by gradual mineralization of collagen.Dentinogenesis is directed by a series of highly controlled biochemicalevents that control the rates of collagen secretion, its maturation intothick fibrils, loss of proteoglycans, mineral formation includinghydroxy apatite crystallization, and growth. The dentin matrix isprimarily composed of collagens (e.g., types I, III and V) as well asother matrix proteins, including, but not limited to, phosphorylated andnonphosphorylated matrix proteins, proteoglycans, growth factors,metalloproteinases, alkaline phosphatase serum derived proteins, andphospholipids. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570). Nostem cells have been identified in mature dentin.

The dental pulp is the tooth's living tissue that respond to pain anddamage and initiates tissue repair. An odontoblast cell layer forms theouter boundary of the pulp and is associated with an underlying networkof dendritic cells. A cell-free zone underlying the odontoblast layer isrich in nerve fibers and blood vessels. Similar to dentin, dental pulpalso differentiates from neural crest-derived ectomesenchyme duringtooth development.

Several sources of stem cells have been identified associated with pulptissue. In immature teeth, apical papilla, the embryonal organresponsible for pulp differentiation, is the source for stem cells ofapical papilla (SCAP). Mature dental pulp is the source of dental pulpstem cells (DPSC) whereas stem cells are also extracted from exfoliateddeciduous teeth (SHED). Additional cells of the dental pulp core thatfunctionin pulpal defense, include, but are not limited to, macrophages,lymphocytes and mast cells. Pulp matrix is composed of collagens (e.g.,types I, III V and VI), but lacks mineralization. Other noncollagenousproteins of the pulp matrix are similar in composition to dentin. Thedental pulp is capable of responding to dentin tissue damage bysecreting new dentin from old odontoblast populations or generation andsecretion of dentin from new secondary odontoblast populations. (Fong etal., 2005, J. Dent. Educ., 69(5): 555-570).

The periodontium consists of tissues supporting the tooth crown,including a nonmineralized periodontal ligament (PDL) sandwiched betweenlayers of mineralized tissues, including the cementum, alveolar bone anddentin. Cementum is a thin mineralized layer covering the dentin.Cementoblasts are cells responsible for cementum matrix secretion andsubsequent mineralization. When cementoblasts become entrapped withincementum matrix, they are termed cementocytes. Cementoblasts areectomesenchymal, being derived from neural crest cells, similar to PDLand alveolar bone. Like bone and dentin, cementum is a collagenousmineralized tissue that hardens upon formation of carbonatedhydroxyapatite. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).

PDL is a space between cementum and alveolar bone. It represents areplacement of the dental follicle region in immature developing teeth.Mature PDL contains mostly periodontal fibroblasts as well as stemcells, known as the periodontal ligament stem cells (PDLSCs). Theimmature dental follicle is also a source of mesenchymal stem cells,known as dental follicle stem cells (DFSCs). (Fong et al., 2005, J.Dent. Educ., 69(5): 555-570).

Table 6 shows the differentiation potential of dental mesenchymal cells.(Ulmer et al., 2010, Schweiz Monatsschr Zahnmed, 120:860-872).

TABLE 6 Differentiation Potential Dental Mesenchymal Stem Cells DPSCSHED PDLSC DFSC SCAP Adipocytes X X X X Cementoblasts X X Chondrocytes XX Dental pulp X Dentin X Endothelocytes X X Musculature X Neuroblasts XNeurons X X Odontoblasts X X X X Osteoblasts X X X X X PDL X ProgenitorsPeriodontium X

Several dental stem cell markers have been identified. Stro-1 and Stro-4are commonly used dental stem cell markers for all dental mesenchymalstem cells. Dental stem cells originating from the neural crest have theneural marker, nestin. An osteoblast marker, osteocalcin, is also usedas a stem cell marker for DPSCs. Similarly, SCAPs express Oct-4, Nanog,SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. (Ulmer et al., 2010, SchweizMonatsschr Zahnmed, 120:860-872).

2.5. Fascial Tissue Compartment

Fascial tissue compartments form a layer of fibrous tissue foundthroughout the body surrounding softer and more delicate organs,including but not limited to muscles, groups of muscles, blood vessels,nerves, etc. Fascial tissue originates from the embryonic mesenchyme.Fasciae form during the development of bones, muscles and vessels fromthe mesodermal layer of the embryo. Fascial tissue can be categorizedinto three types depending on location: (1) superficial fascial tissue,which is found beneath the integument throughout the body, usuallyblending with the reticular layer of the dermis; (2) deep fascial tissuecomprising dense fibroareolar connective tissue surrounding muscles,bones, nerves and blood vessels; and (3) visceral or subserous fascia,which suspends organs within their cavities and wraps them in layers ofconnective tissue membranes. (Chapter IV. Myology, Section 3. Tendons,Aponeuroses, and Fasciae, Gray's Anatomy of the Human Body, 20^(th)Edition, Re-edited by Lewis, W. H., Lea & Febiger, Philadelphia, 1918,Bartleby.com, New York, 2000).

The fibroareolar connective tissue of fascia comprises four kinds ofcells: (1) flattened lamellar cells, which may be branched or unbranched(branched lamellar cells contain clear cytoplasm and oval nuclei andproject multidirectional processes that may unite to form an opennetwork, such as in the cornea; unbranched lamellar cells are joined endto end. (2) Clasmatocytes, which are large irregular vacuolated orgranulated cells with oval nuclei. (3) Granule cells, which are ovoid orspherical in shape. (4) Plasma cells of Waldeyer, usually spheroidal,characterized by vacuolated protoplasm.

2.5. Ligament Tissue Compartment

The term “ligaments” as used herein refers to dense regular connectivetissue comprising attenuated collagenous fibers that connect bones atjoints. Ligament ECM is composed of type I and type III collagenstogether with other proteoglycans and glycoproteins. Mesenchymal stemcells have been found in the human anterior cruciate ligament thatexhibit multilineage differentiation potential, like bone-derivedmesenchymal stem cells. (Cheng et al., 2010, Tissue Engg. A,16(7):2237-2253).

2.6. Synovial Tissue Compartment

The synovial membrane is composed of fibrous connective tissue and linesthe joint cavity of synovial joints. It is made up of a layer ofmacrophage (type A) and fibroblast-like (type B) synoviocytes and aloose sublining tissue. Synovial fluid is secreted by synovial cellslining the synovial membrane in the joint capsule. It is a viscid,mucoalbuminous fluid, rich in hyaluronic acid. It acts as a lubricatingfluid, facilitating the smooth gliding of the articular surface.Functional mesenchymal stem cell niches have been identified as residentto synovial lining and subsynovial tissue. These cells are positive forthe artificial nucleoside, iododeoxyuridine (IdU) as well as MSC markerssuch as PDGFRα, p75 and CD44 and have chondrogenic potential. (Kurth etal., Arthritis Rheum., 2011, 63(5): 1289-1300). Synovial fluid-derivedMSCs have also been identified, and these have higher chondrogenicpotential as compared to bone marrow-derived and adipogenic MSCs. (Kogaet al., 2008, Cell Tissue Res., 333: 207-215). Synovial MSCs and MPCshave been shown to prevent degeneration due to intervertebral discdisease (IVD) and to be useful for cartilage tissue engineering.(Miyamoto et al., 2010, Arthritis Res. Ther., 12: R206-218; Lee et al.,2010, Tissue Engg. A, 16(1): 317-325).

2.7. Tendon Tissue Compartment

Tendons are specialized connective tissue compartments that connect boneto muscle. Tendon cells are embedded amongst a parallel group ofcollagenous fibers that secrete a unique ECM containing collagens, largeproteoglycans, and small leucine rich proteoglycans that function aslubricators and organizers of collagen fibril assembly. A unique tendonstem/progenitor cell (TSPC) niche has been identified amongst theparallel collagen fibrils surrounded by ECM. The TSPCs exhibitosteogenic and adipogenic potential. Biglycan and fibromodulin are keytendon ECM components that direct TSPC fate through BMP signaling. TheseTSPCs are positive for bone marrow derived stem cell markers such asStro-1, CD146, CD90 and CD44 but not for CD18. TSPCs do not expresshematopoietic markers, such as CD34, CD45 and CD117, or the endothelialmarker CD106. (Bi et al., 2007, Nat. Med., 13(10): 1219-1227).

2.8. Vasculature Tissue Compartment

The vascular wall is made of three concentric zones with distinctcellular composition, all mesodermal in origin: the tunica intima,containing predominantly mature differentiated endothelial cells (EC),the tunica media, containing mature and differentiated smooth musclecells, and the tunica adventitia, containing mature fibroblasts. (Tilkiet al., 2009, Trends Mol. Med. 15(11): 501-509). Endothelial progenitorcells (EPCs), meaning cells that exhibit clonal expression, stemnesscharacteristics, adherence to matrix molecules and an ability todifferentiate into endothelial cells (ECs) have been implicated in theformation of new blood vessels through angiogenesis and postnatalvasculogenesis. EPCs have many characteristic cell surface markers,including, but not limited to, CD34, AC133, KDR (VEGFR-2), Tie-2 andligand for UEA-1 lectin. (Tilki et al., 2009, Trends Mol. Med. 15(11):501-509; Melero-Martin and Dudley, 2011, Stem Cells, 29: 163-168;Pascilli et al., 2008, Exp. Cell Res., 315: 901-914).

EPC niches have been identified in the bone-marrow, peripheral cordblood and vascular wall matrix. Bone-marrow derived and cord blood EPCsessentially may be proangiogenic hematopoietic progenitor cells (HPCs),circulating in the blood and committed to myeloid lineage. (Tilki etal., 2009, Trends Mol. Med. 15(11): 501-509). The vascular wall stem andprogenitor cells (VW-EPCs) reside in distinct zones of the vessel wallwithin subendothelial space, known as avasculogenic zone, within thevascular adventitia, forming vascular wall-specific niches. Fetal andadult arterial and venous blood vessel walls have also been found toharbor resident niches for a variety of stem and progenitor cells, suchas EPCs, smooth muscle progenitors, HSCs, MSCs, mesangial cellscoexpressing myogenic and endothelial markers, neural stem cells (NSCs),etc. (Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509). The VW-EPCsare CD34(+)VEGFR-2(+)Tie-2(+)CD31(−)CD144(−). Proliferating anddifferentiating VW-EPCs become CD144(+).

During embryogenesis, there is evidence of the existence of ahemangioblast (giving rise to endothelial and hematopoietic cells) andhemogenic endothelium, originating from precursors resident in thevascular wall. However, whether adult VW also contains ancestralprogenitor hemangioblasts giving rise to both VW-EPCs as well as VW-HSCsis not known. Vascular wall also contains resident pericyte-like cellsin the subendothelial spaces. These pericyte-like cells serve as acellular reservoir for VW-MSCs, which can differentiate into colonieswith adipogenic, osteogenic and chondrgenic markers. (Tilki et al.,2009, Trends Mol. Med. 15(11): 501-509).

3. Cells of the Epithelial Tissue Compartment

3.1. Placental Tissue Matrix

The placenta is considered one of the most important sources of stemcells, and has been studied extensively. It fulfills two main desiderataof cell therapy: a source of a high as possible number of cells and theuse of non-invasive methods for their harvesting. Their highimmunological tolerance supports their use as an adequate source in celltherapy (Mihu, C. et al., 2008, Romanian Journal of Morphology andEmbryology, 2008, 49(4):441-446).

The fetal adnexa is composed of the placenta, fetal membranes, andumbilical cord. The term placenta is discoid in shape with a diameter of15-20 cm and a thickness of 2-3 cm. The fetal membranes, amnion andchorion, which enclose the fetus in the amniotic cavity, and theendometrial decidua extend from the margins of the chorionic disc. Thechorionic plate is a multilayered structure that faces the amnioticcavity. It consists of two different structures: the amniotic membrane(composed of epithelium, compact layer, amniotic mesoderm, and spongylayer) and the chorion (composed of mesenchyme and a region ofextravillous proliferating trophoblast cells interposed in varyingamounts of Langhans fibrinoid, either covered or not bysyncytiotrophoblast).

Villi originate from the chorionic plate and anchor the placenta throughthe trophoblast of the basal plate and maternal endometrium. From thematernal side, protrusions of the basal plate within the chorionic villiproduce the placental septa, which divide the parenchyma into irregularcotyledons (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).

Some villi anchor the placenta to the basal plate, whereas othersterminate freely in the intervillous space. Chorionic villi present withdifferent functions and structure. In the term placenta, the stem villishow an inner core of fetal vessels with a distinct muscular wall andconnective tissue consisting of fibroblasts, myofibroblasts, anddispersed tissue macrophages (Hofbauer cells). Mature intermediate villiand term villi are composed of capillary vessels and thin mesenchyme. Abasement membrane separates the stromal core from an uninterruptedmultinucleated layer, called the syncytiotrophoblast. Between thesyncytiotrophoblast and its basement membrane are single or aggregatedLanghans cytotrophoblastic cells, commonly called cytotrophoblast cells(Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).

Four regions of fetal placenta can be distinguished: an amnioticepithelial region, an amniotic mesenchymal region, a chorionicmesenchymal region, and a chorionic trophoblastic region.

Amniotic Membrane

Fetal membranes continue from the edge of the placenta and enclose theamniotic fluid and the fetus. The amnion is a thin, avascular membranecomposed of an inner epithelial layer and an outer layer of connectivetissue that, and is contiguous, over the umbilical cord, with the fetalskin. The amniotic epithelium (AE) is an uninterrupted, single layer offlat, cuboidal and columnar epithelial cells in contact with amnioticfluid. It is attached to a distinct basal lamina that is, in turn,connected to the amniotic mesoderm (AM). In the amniotic mesodermclosest to the epithelium, an acellular compact layer isdistinguishable, composed of collagens I and III and fibronectin. Deeperin the AM, a network of dispersed fibroblast-like mesenchymal cells andrare macrophages are observed. It has been reported that the mesenchymallayer of amnion indeed contains two subfractions, one having amesenchymal phenotype, also known as amniotic mesenchymal stromal cells,and the second containing monocyte-like cells.

Chorionic Membrane

A spongy layer of loosely arranged collagen fibers separates theamniotic and chorionic mesoderm. The chorionic membrane (chorion leave)consists of mesodermal and trophoblastic regions. Chorionic and amnioticmesoderm are similar in composition. A large and incomplete basal laminaseparates the chorionic mesoderm from the extravillous trophoblastcells. The latter, similar to trophoblast cells present in the basalplate, are dispersed within the fibrinoid layer and expressimmunohistochemical markers of proliferation. The Langhans fibrinoidlayer usually increases during pregnancy and is composed of twodifferent types of fibrinoid: a matrix type on the inner side (morecompact) and a fibrin type on the outer side (more reticulate). At theedge of the placenta and in the basal plate, the trophoblastinterdigitates extensively with the decidua (Cunningham, F. et al., Theplacenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton andLange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of thehuman placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297).

Amnion-Derived Stem Cells

The amniotic membrane itself contains multipotent cells that are able todifferentiate in the various layers. Studies have reported theirpotential in neural and glial cells, cardiac repair and also hepatocytecells. Studies have shown that human amniotic epithelial cells expressstem cell markers and have the ability to differentiate toward all threegerm layers. These properties, the ease of isolation of the cells, andthe availability of placenta, make amnionic membrane a useful andnoncontroversial source of cells for transplantation and regenerativemedicine.

Amniotic epithelial cells can be isolated from the amniotic membrane byseveral methods that are known in the art. According to one such method,the aminiotic membrane is stripped from the underlying chorion anddigested with trypsin or other digestive enzymes. The isolated cellsreadily attach to plastic or basement membrane-coated culture dishes.Culture is established commonly in a simple medium such as Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 5%-10% serum andepidermal growth factor (EGF), in which the cells proliferate robustlyand display typical cuboidal epithelial morphology. Normally, 2-6passages are possible before proliferation ceases. Amniotic epithelialcells do not proliferate well at low densities.

Amniotic membrane contains epithelial cells with different surfacemarkers, suggesting some heterogeneity of phenotype. Immediately afterisolation, human amniotic epithelial cells express very low levels ofhuman leukocyte antigen (HLA)-A, B, C; however, by passage 2,significant levels are observed. Additional cell surface antigens onhuman amniotic epithelial cells include, but are not limited to,ATP-binding cassette transporter G2 (ABCG2/BCRP), CD9, CD24, E-cadherin,integrins α6 and β1, c-met (hepatocyte growth factor receptor),stage-specific embryonic antigens (SSEAs) 3 and 4, and tumor rejectionantigens 1-60 and 1-81. Surface markers thought to be absent on humanamniotic epithelial cells include SSEA-1, CD34, and CD133, whereas othermarkers, such as CD117 (c-kit) and CCR4 (CC chemokine receptor), areeither negative or may be expressed on some cells at very low levels.Although initial cell isolates express very low levels of CD90 (Thy-1),the expression of this antigen increases rapidly in culture (Miki, T. etal., Stem Cells, 2005, 23: 1549-1559; Miki, T. et al., Stem Cells, 2006,2: 133-142).

In addition to surface markers, human amniotic epithelial cells expressmolecular markers of pluripotent stem cells, including octamer-bindingprotein 4 (OCT-4) SRY-related HMG-box gene 2 (SOX-2), and Nanog (Miki,T. et al., Stem Cells, 2005, 23: 1549-1559). Previous studies also haveshown that human amnion cells in xenogeneic, chimeric aggregates, whichcontain mouse embryonic stem cells, can differentiate into all threegerm layers and that cultured human amniotic epithelial cells expressneural and glial markers, and can synthesize and release acetylcholine,cateholamines, and dopamine. Hepatic differentiation of human amnioticepithelial cells also has been reported. Studies have reported thatcultured human amniotic epithelial cells produce albumin andα-fetroprotein and that albumin and α-fetroprotein-positivehepatocyte-like cells could be identified integrated into hepaticparenchyma following transplantation of human amniotic epithelial cellsinto the livers of severe combined inmmunodeficiency (SCID) mice. Thehepatic potential of human amniotic epithelial cells was confirmed andextended, whereby in addition to albumin and α-fetroprotein production,other hepatic functions, such as glycogen storage and expression ofliver-enriched transcription factors, such as hepatocyte nuclear factor(HNF) 3γ and HNF4α, CCAAT/enhancer-binding protein (CEBP α and β), andseveral of the drug metabolizing genes (cytochrome P450) weredemonstrated. The wide range of hepatic genes and functions identifiedin human amniotic epithelial cells has suggested that these cells may beuseful for liver-directed cell therapy (Parolini, O. et al., 2008, StemCell, 2008, 26:300-311).

Differentiation of human amniotic epithelial cells to another endodermaltissue, pancreas, also has been reported. For example, it was shown thathuman amniotic epithelial cells cultured for 2-4 weeks in the presenceof nicotinamide to induce pancreatic differentiation, expressed insulin.Subsequent transplantation of the insulin-expressing human amnioticepithelial cells corrected the hyperglycemia of streptozotocin-induceddiabetic mice. In the same setting, human amniotic mesenchymal stromalcells were ineffective, suggesting that human amniotic epithelial cells,but not human amniotic mesenchymal stromal cells, were capable ofacquiring β-cell fate (Parolini, O. et al., 2008, Stem Cell, 2008,26:300-311).

Mesenchymal Stromal Cells from Amnion and Chorion: hAMSC and hCMSC

Human amniotic mesenchymal cells (hAMSC) and human chorionic mesenchymalcells (hCMSC) are thought to be derived from extraembryonic mesoderm.hAMSC and hCMSC can be isolated from first-, second-, andthird-trimester mesoderm of amnion and chorion, respectively. For hAMSC,isolations are usually performed with term amnion dissected from thedeflected part of the fetal membranes to minimize the presence ofmaternal cells. For example, homogenous hAMSC populations can beobtained by a two-step procedure, whereby: minced amnion tissue istreated with trypsin to remove hAEC and the remaining mesenchymal cellsare then released by digestion (e.g., with collagenase or collagenaseand DNase). The yield from term amnion is about 1 million hAMSC and10-fold more hAEC per gram of tissue (Casey, M. and MacDonald P., BiolReprod, 1996, 55: 1253-1260).

hCMSCs are isolated from both first- and third-trimester chorion aftermechanical and enzymatic removal of the trophoblastic layer withdispase. Chorionic mesodermal tissue is then digested (e.g., withcollagenase or collagenase plus DNase). Mesenchymal cells also have beenisolated from chorionic fetal villi through explant culture, althoughmaternal contamination is more likely (Zhang, X., et al., BiochemBiophys Res Commun, 2006, 340: 944-952; Soncini, M. et al., J Tissue EngRegen Med, 2007, 1:296-305; Zhang et al., Biochem Biophys Res Commun,2006, 351: 853-859).

The surface marker profile of cultured hAMSC and hCMSC, and mesenchymalstromal cells (MSC) from adult bone marrow are similar. All expresstypical mesenchymal markers (Table 7) but are negative for hematopoietic(CD34 and CD45) and monocytic markers (CD14). Surface expression ofSSEA-3 and SSEA-4 and RNA for OCT-4 has been reported (Wei J. et al.,Cell Transplant, 2003, 12: 545-552; Wolbank, S. et al., Tissue Eng,2007, 13: 1173-1183; Alviano, F. et al., BMC Dev Biol, 2007, 7: 11;Zhao, P. et al, Transplantation, 2005, 79: 528-535). Both first- andthird trimester hAMSC and hCMSC express low levels of HLA-A, B, C butnot HLA-DR, indicating an immunoprivileged status (Portmann-Lanz, C. etal, Am J Obstet Gynecol, 2006, 194: 664-673; Wolbank, S. et al., TissueEng, 2007, 13: 1173-1183).

Table 7 provides surface antigen expression profile at passages 2-4 foramniotic mesencymal stromal and human chorionic mesenchymal stromal stemcells.

TABLE 7 Specific surface antigen expression for aminiotic mesenchymalstromal cells and human chorionic mesenchymal stromal cells Positive(≧95%) Negative(≦2%) CD90 CD45 CD73 CD34 CD105 HLA-DR

Both hAMSCs and hCMSCs differentiate toward “classic” mesodermallineages (osteogenic, chondrogenic, and adipogenic) and differentiationof hAMSC to all three germ layers-ectoderm (neural), mesoderm (skeletalmuscle, cardiomyocytic and endothelial), and endoderm (pancreatic) wasreported (Int'Anker, P. et al., Stem Cells, 2004, 22: 1338-1345;Portmann-Lanz, C. et al, Am J Obstet Gynecol, 2006, 194: 664-673;Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Soncini, M. et al.,J Tissue Eng Regen Med, 2007, 1:296-305; Alviano, F., BMC Dev Biol,2007, 7: 11).

Human amniotic and chorionic cells successfully and persistently engraftin multiple organs and tissues in vivo. Human chimerism detection inbrain, lung, bone marrow, thymus, spleen, kidney, and liver after eitherintraperitoneal or intravenous transplantation of human amnion andchorion cells into neonatal swine and rats was indeed indicative of anactive migration consistent with the expression of adhesion andmigration molecules (L-selectin, VLA-5, CD29, and P-selectin ligand 1),as well as cellular matrix proteinase (MMP-2 and MMP-9) (Bailo, M. etal., Transplantation, 2004, 78:1439-1448).

Umbilical Cord

Two types of umbilical stem cells can be found, namely hematopoieticstem cells (UC-HS) and mesenchymal stem cells, which in turn can befound in umbilical cord blood (UC-MS) or in Wharton's jelly (UC-MM). Theblood of the umbilical cord has long been in the focus of attention ofresearchers as an important source of stem cells for transplantation,for several reasons: (1) it contains a higher number of primitivehematopoietic stem cells (HSC) per volume unit, which proliferate morerapidly, than bone marrow; (2) there is a lower risk of rejection aftertransplantation; (3) transplantation does not require a perfect HLAantigen match (unlike in the case of bone marrow); (4) UC blood hasalready been successfully used in the treatment of inborn metabolicerrors; and (5) there is no need for a new technology for collection andstorage of the mononuclear cells from UC blood, since such methods arelong established.

Umbilical cord (UC) vessels and the surrounding mesenchyma (includingthe connective tissue known as Wharton's jelly) derive from theembryonic and/or extraembryonic mesodermis. Thus, these tissues, as wellas the primitive germ cells, are differentiated from the proximalepiblast, at the time of formation of the primitive line of the embryo,containing MSC and even some cells with pluripotent potential. The UCmatrix material is speculated to be derived from a primitive mesenchyma,which is in a transition state towards the adult bone marrow mesenchyma(Mihu, C. et al., 2008, Romanian Journal of Morphology and Embryology,2008, 49(4):441-446).

The blood from the placenta and the umbilical cord is relatively easy tocollect in usual blood donation bags, which contain anticoagulantsubstances. Mononuclear cells are separated by centrifugation on Ficollgradient, from which the two stem cell populations will be separated:(1) hematopoietic stem cells (HSC), which express certain characteristicmarkers (CD34, CD133); and (2) mesenchymal stem cells (MSC) that adhereto the culture surface under certain conditions (e.g., modified McCoymedium and lining of vessels with Fetal Bovine Serum (FBS) or Fetal CalfSerum (FCS)). (Munn, D. et al., Science, 1998, 281: 1191-1193; Munn, D.et al., J Exp Med, 1999, 189: 1363-1372). Umbilical cord blood MSCs(UC-MS) can produce cytokines, which facilitate grafting in the donorand in vitro HSC survival compared to bone marrow MSC. (Zhang, X et al.,Biochem Biophys Res Commun, 2006, 351: 853-859).

MSCs from the umbilical cord matrix (UC-MM) are obtained by differentculture methods depending on the source of cells, e.g., MSCs from theconnective matrix, from subendothelial cells from the umbilical vein oreven from whole umbilical cord explant. They are generally well culturedin DMEM medium, supplemented with various nutritional and growthfactors; in certain cases prior treatment of vessels with hyaluronicacid has proved beneficial (Baban, B. et al., J Reprod Immunol, 2004,61: 67-77).

3.2. Lung

The lungs, which are paired organs that fill up the thoracic cavity,constitute an efficient air-blood gaseous exchange mechanism,accomplished by the passage of air from the mouth or nose, sequentiallythrough an oropharynx, nasopharynx, a larynx, a trachea and finallythrough a progressively subdividing system of bronchi and bronchiolesuntil it finally reaches alveoli where the air-blood gaseous exchangetakes place. A resident niche with characteristic multipotent stem cellswith c-kit positive surface profiles recently has been identifiedlocalized in small bronchioles alveoli. These stem cells express thetranscription factors, Nanog, Oct3/4, Sox2 and K1f4, that governpluripotency in embryonic stem cells. (Kajstura, J. et al., 2011, NewEngl. J. Med., 364(19):1795-1806)).

3.2. Mammary

The mammary gland is a hormone sensitive bilayered epithelial organcomprising an inner luminal epithelial layer and an outer myoepitheliallayer surrounded by a basement membrane in a stromal fat pad. Mammarystem cells with myoepithelial potential have been identified in theirniches in the terminal ducts of mammary gland. (LaBarge, 2007, Stem CellRev., 3(2): 137-146).

3.3. Skin

The skin functions as the primary barrier imparting protection fromenvironmental insults. Skin is composed of an outer epidermis and innerdermis separated by a basement membrane (BM), rich in ECM and growthfactors. The BM of the epidermal-dermal junction is composed ofcollagens (e.g., type IV and XVII), laminins, nidogen, fibronectin andproteoglycans that provide storage sites for growth factors andnutrients supporting the proliferation and adhesion of epidermalkeratinocytes.

The epidermis is a solid epithelial tissue comprising keratinocytes thatare linked to each other via cellular junctions, such as desmosomes.Keratinocytes are organized into distinct layers, comprising the stratumcorneum, stratum granulosum, stratum spinosum and stratum basale. Theepidermal matrix is made up of hyaluronan and other proteoglycans,including but not limited to, desmosealin, glycipans, versican,perlecan, and syndecans. (Sandjeu and Haftek, 2009, J. Physiol.Pharmacol. 60 (S4): 23-30). Epidermal desmosomes are multimericcomplexes of transmembrance glycoprotein and cytosolic proteins with thekeratin cytoskeleton. Desmosal proteins of the epidermis predominantlybelong to the cadherin, Armadillo and plakin superfamilies.

The underlying dermis is connective tissue comprised primarily offibroblasts with occasional inflammatory cells. Embedded within thedermis are also epidermal appendages, such as hair follicles andsebaceous glands, as well as nerves and cutaneous vasculature. Thedermal ECM is essentially made of type I, III and V collagens andelastin together with noncollagenous components such as glycoproteins,proteoglycans, GAGs, cytokines and growth factors. Dermal collagens helpmediate fibroblast-matrix interactions through a number of cell surfacereceptors and proteoglycans, such as β1-integrins. (Hodde and Johnson,2007, Am. J. Clin. Dermatol. 8(2): 61-66).

During embryonic development, the epidermis originates from theectoderm, while the dermis differentiates from the mesoderm. Followinggastrulation, as mesenchymal stem cells of mesodermal origin populatethe skin, they send signals to the single epidermal layer for initiationof epidermal stratification and direct the positioning of outgrowths ofepidermal appendages, such as the hair follicles and sebaceous glands.Along with the mesenchyme, the basal layer of the epidermis organizesinto a basement membrane that is rich in ECM proteins and growthfactors. A number of different signaling pathways have been implicatedin skin morphogenesis, including but not limited to Notch, Wnt, mitogenactivated protein kinase (MAPK), nuclear factor-κB (NF-κB),transcriptional regulator, p63, the AP2 family of transcription factors,CCAAT/enhancer binding protein (C/EBP) transcriptional regulators,interferon regulatory 6 (URF6), grainyhead-like 3 (GRHL3) andKruppel-like factor (KLF4). (Blanpain and Fuchs, 2009, Nat. Rev. Mol.Cell. Biol., 10(3): 207-217).

Adult skin undergoes constant cellular turnover whereby dead skin cellsare shed and new cells are regenerated and replaced, by a process knownas skin homeostasis. Several stem cell niches with distinct surfacemarker profiles and differentiation potentials have been identified.These include, but are not limited to, epidermal stem cells ofinterfollicular epidermis; bulge stem cells and epithelial stem cells ofthe hair follicle, dermal stem cells (e.g., multipotent dermal cells,skin-derived progenitor cells, dermis-derived multipotent stem cells andfibrocytes), dermal papilla stem cells, and sebaceous gland stem cells.Collectively, these skin stem cell niches partake in maintaining skinhomeostasis with the help of growth factors and cytokines (Zouboulis etal., 2008, Exp. Gerontol. 43: 986-997; Blanpain, 2010, Nature, 464:686-687).

4. Cells of the Muscular Tissue Compartment

The muscular tissue compartments are comprised of contractile muscletissue. These can be of three kinds: skeletal muscle associated with theskeletal system; cardiac muscle associated with the heart; and smoothmuscle associated with the vasculature and gastrointestinal tract.Skeletal muscle tissue fibers are striated and are voluntary infunction. Cardiac muscle fibers have characteristic intercalated discsand are involuntary in function. Smooth muscle tissue is comprised ofspindle shaped cells and is involuntary in function.

Skeletal muscles are composed of a population of quiescent myogenicprecursor cells known as satellite cells with muscle regenerating andself-renewal properties, as well as a population of multipotentmuscle-derived stem cells (MDSC) with multilineage differentiationpotential, such as mesodermal lineages including, but not limited to,myogenic lineages, adipogenic lineages, osteogenic lineages,chondrogenic lineages, endothelial and hematopoetic lineages, andectodermal lineages, including not limited to neuron-like cells. (Xu etal., 2010, Cell Tissue Res., 340: 549-567).

Skeletal muscle satellite cells are quiescent mononucleated cells thatare resident in the muscle fiber membrane, beneath the basal laminaforming distinct stem cell niches. Similar to other stem cell niches,the skeletal muscle satellite cell niche is a dynamic structure, capableof altering between inactive (quiescent) and activated states inresponse to external signals. Once activated, satellite cells have thepotential to proliferate, expand and differentiate along the myogeniclineage. The basal lamina, which serves to separate individual skeletalmuscle fibers, known as myofibers, and their associated satellite celland stem cell niches, from the cells of the interstitium, is rich incollagen type IV, perlecan, laminin, entactin, fibronectin and severalother glycoproteins and proteoglycans, that may function as receptors togrowth factors effectuating their activation by extracellular processingand modifications. In addition to these interactions provided by theECM, neighboring cells, such as endothelial cells and multipotent stemcells derived from blood vessels, such as pericytes and mesoangioblasts,or neural components, all have the potential of affecting the nichemicroenvironment. (Gopinath et al., 2008, Aging Cell, 7: 590-598).

Endogenous cardiac stem cells have also been identified in cardiac stemcell niches. (Mazhari and Hare, 2007, Nat. Clin. Pract. Cardiovasc.Med., 4(S1): S21-S26).

Vascular smooth muscle cells are derived from embryonic cardiac neuralcrest stem cells, as well as proepicardial cells and endothelialprogenitor cells. Smooth muscle differentiation is dependent on acombination of factors, including but not limited to Pax3, Tbx1, FoxC1and serum response factor, interacting with microenvironment componentsof the ECM, such as BMPs, Wnts, endothelin (ET)-1, and FGF8. In theadult, vascular smooth muscle cells undergo constant degeneration,repair and regeneration by the concerted efforts of both multipotentbone-derived mesenchymal cells as well as smooth muscle stem cellsresident within vascular smooth muscle tissue. (Hirschi and Majesky,2004, The Anatomical Record, Part A, 276A: 22-33).

5. Cells of the Neural Tissue Compartment

The neural tissue compartments are comprised of neurons and theneuroglia, embedded with the neural matrix. Neural tissue is ectodermalin origin, derived from the embryonic neural plate. Neural tissue isprimarily located within the brain, spinal cord and nerves.

Resident neural stem cell niches have been identified in the adultmammalian brain, restricted to the subventricular zone as well as to thelateral ventricle and dentate gyms subgranular zone of the hippocampus.Astrocytes, which are star-shaped nerve cells, serve as both neural stemcells as well as supporting niche cells secreting essential growthfactors that provide support for neurogenesis and vasculogenesis. Thebasal lamina and associated vasculogenesis are essential components ofthe niche. Embryonic molecular factors and signals persist within theneural stem cell niches and play critical role in neurogenesis. Neuralstem cells have VEGFR2, doublecortin and Lex (CD15) markers. Majorsignaling pathways implicated in neurogenesis include but are notlimited to Notch, Eph/ephrins, Shh, and BMPs. (Alvarez-Buylla and Lim,2004, Neuron, 41: 683-686).

6. Grafts—Grafts and Graft Rejection

A graft is a tissue or organ used for transplantation to a patient. Acommon strategy employed in tissue engineering involves the seeding ofdecellularized natural ECM or synthetic scaffolds with a variety ofdifferent stem or progenitor cells that are capable of regeneration(see, for example, Flynn and Woodhouse, 2008, Organogenesis, 4(4):228-235; Uriel et al., 2008, Biomaterials, 29: 3712-3719; Flynn, 2010,Biomaterials, 31: 4715-4724; Choi et al., Tissue Engg. C., 16(3):387-396; Brown et al., 2011, Tissue Engg. C., 17(4): 411-421; Cheng etal., 2009, Tissue Engg. A, 15(2): 231-241; Li et al., 2011,Biomaterials, doi:10:1016/j.biomaterials.2011.03.008; Butler et al.,2003, Connective Tissue Research, 44(S1): 171-178); Mercuri et al., J.Biomed. Mater. Res. A., 96(2): 422-435); Olson et al., 2011, Chonnam.Med. J. 47:1-13).

Transplanted grafts may be rejected by the recipient host via anorchestrated immune response against the histocompatibility antigensexpressed by the grafted tissue, which the recipient host may see asforeign. Effectors primarily responsible for such rejections includetype 1 helper CD4+ cells, cytotoxic CD8+ cells and antibodies.Alternative mechanisms of rejection include the involvement of type 2helper CD4+ cells, memory CD8+ cells, and cells that belong to theinnate immune system, such as natural killer cells, eosinophils, andneutrophils. In addition, local inflammation associated with rejectionis tightly regulated at the graft level by regulatory T cells and mastcells.

Implants

Patients suffering from affected or injured organs may be treated withorgan transplantation. However, current methods of organ transplantationare faced with challenges due, in part to the need to suppress immunerejection of the transplanted organ. Most methods rely on the use ofimmunesuppressive drugs that are associated with unwanted side effects.

It is estimated that more than one million patients need to be treatedsurgically for skeletal afflictions every year due to bony defectscreated during tumor surgery or caused by trauma, congenital skeletalabnormalities, fracture, scoliosis, spinal arthrodesis, or joint andtooth replacement. Surgical treatments, however, are not alwayseffective to address these problems because of inadequate local boneconditions and impaired bone healing. For example, complicated fracturesmay fail to heal, resulting in delayed unions (a bone fracture that istaking an exceptionally long amount of time to heal) or non-unions(absence of healing in a fracture). In addition, the treatment of bonetumors or congenital syndromes often requires the artificial creation oflarge bony defects, which need to be filled, demanding suitable andbiocompatible substitutes for bone grafts.

Bone healing around implants involves the activation of a sequence ofosteogenic, vascular, and immunological events that are similar to thoseoccurring during bone healing. Various cell types, growth factors andcytokines are involved and interact throughout the stages ofosteointegration, including inflammation, vascularization, boneformation, and ultimately bone remodeling.

Bone Grafts

Fresh autologous bone grafts for the treatment of an osseous defect orfracture in a patient are derived from bone marrow freshly harvestedfrom the iliac crest (the thick curved upper border of the ilium, themost prominent bone on the pelvis) and combined with other materialsincluding osteoconductive substrates. Complications associated withautologous harvest include donor site morbidity as high as 25%,infection, malformation, pain, and loss of function.

Bone Matrix with Mesenchymal Stem Cells

Attempts have been made to repair osseous defects by implanting a bonematrix comprising autologous or allogeneic mesenchymal stem cells(MSCs). MSCs are considered immunologically neutral, meaning that themesenchymal stem cells from the donor need not be tissue-matched to therecipient, thus allowing MSCs to be used effectively in allogeneicgrafts. In addition, culture-expanded allogeneic MSCs have beenimplanted either directly or combined with a matrix, such as agelatin-based or collagen-based matrix, or a bone matrix, in order tosupport differentiation of the MSCs in vivo.

In other instances, MSCs have been combined with a bone matrix fromwhich bone marrow has been removed in order to remove undesirable cells,and the matrix then seeded with culture-expanded MSCs. Such compositionsthen are cryopreserved under standard cryopreservation procedures forlater use. However, this method is not ideal for several reasons. First,because the MSCs have been removed from the original stem cell niche andseeded onto a new bone matrix, the MSCs in such a composition are notwell-attached to the bone matrix and become merely suspended in thecryopreservation solution. As a result, many active cells can be lostduring the process of removing the cryopreservation solution beforetransplantation into a subject. Secondly, since the cells are notattached to the stem cell niche or lacunae to which they were originallyattached and in which they were nurtured, the expandability andosteogenic potential of the cells may be affected negatively by theseparation and seeding procedures.

Tissue-derived implant materials replicate the biological and mechanicalfunction of naturally occurring extracellular matrix found in bodytissues. Such tissue-derived matrices provide the necessary support onwhich cells can adhere to, migrate and expand and allow the influx andefflux of cells, such as stem cells and progenitor cells, and otherfactors, such as growth factors and cytokines, capable of inducing andsupporting growth and tissue repair.

A new approach to prepare and transplant an allograft is presentedherein in which biological contents of a matrix are preserved such thatfactors and biologically active cells with the potential todifferentiate into adult tissue cells are attached in situ, andundesirable cells are removed. An alternative approach, in whichbiologically active cells with the potential to differentiate into adulttissue cells, growth-inductive and growth-conductive factors are addedback to a tissue derived matrix also is presented. Such approaches wouldallow faster regeneration of tissue in transplanted individuals.

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides atissue-derived implant comprising: (a) at least one tissue-derivedgrowth conductive matrix from which unwanted cells have been removed;and (b) at least one viable population of nonexpanded tissuegenic cellsadherent to and resident in an endogenous milieu of the growthconductive matrix.

According to one embodiment, the implant comprises a plurality of piecesof the growth conductive matrix comprising a circular shape, a squareshape, a polygonal shape, a rectangular shape, a triangular shape, anoctagonal shape, an irregular shape, an elongated shape (e.g., as afiber), an amorphous shape, or a combination thereof. According toanother embodiment, the growth conductive matrix exists in a pluralityof pieces ranging from about 10 μm to about 20 cm in length. Accordingto another embodiment, the plurality of pieces of the growth conductivematrix comprises a block form, a dowel form, a powder form, a slurryform, a paste form, a three-dimensional form, a sheet form, or acombination thereof. According to another embodiment, the growthconductive matrix is derived from a mammalian tissue. According toanother embodiment, the growth conductive matrix is derived from atissue selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, a fascialtissue, a gastrointestinal tissue, a growth plate tissue, anintervertebral disc tissue, an intestinal mucosal tissue, an intestinalserosal tissue, a ligament tissue, a liver tissue, a lung tissue, amammary tissue, a meniscal tissue, a muscle tissue, a nerve tissue, anovarian tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a placental tissue, a skintissue, a spleen tissue, a stomach tissue, a synovial tissue, a tendontissue, a testes tissue, an umbilical cord tissue, a urological tissue,a vascular tissue, a vein tissue, and a combination thereof. Accordingto another embodiment, the tissue is derived from a human donor.According to another embodiment, the human donor is a cadaveric donor.According to another embodiment, the human donor is a living donor.According to another embodiment, the tissuegenic cells are present inthe growth conductive matrix at a relative frequency substantiallysimilar to the relative frequency of the tissuegenic cells found invivo. According to another embodiment, the implant further comprises atleast one growth-inductive component. According to another embodiment,the growth-inductive component is tissue-derived. According to anotherembodiment, the growth-inductive component comprises demineralized bonematrix, which may have any of the shapes or dimensions recited hereinfor growth-conductive matrix, and which may be derived from corticalbone. According to another embodiment, the tissue-derivedgrowth-inductive component originates from a component of thetissue-derived growth-inductive component other than cells. According toanother embodiment, the growth-conductive component comprises a growthmedium derived from expanded tissuegenic cells. According to anotherembodiment, the tissuegenic cells adherent to and resident in anendogenous milieu of the growth conductive matrix secrete the at leastone growth-inductive component. According to another embodiment, the atleast one growth-inductive component comprises at least one cytokineAccording to another embodiment, the at least one growth-inductivecomponent comprises at least one growth factor. According to anotherembodiment, the least one growth factor is selected from the groupconsisting of fibroblast growth factor-2 (FGF-2), fibroblast growthfactor-5 (FGF-5), insulin-like growth factor 1 (IGF-1), neural epidermalgrowth-factor-like 1 (Nel-like 1, NELL1), transforming growth factorbeta (TGF-β), a bone morphogenic protein, bone morphogenic protein-2(BMP-2), bone morphogenic protein-7 (BMP-7), platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF), and acombination thereof. According to another embodiment, thegrowth-conductive matrix and the tissuegenic cells are derived from abone tissue. According to another embodiment, the bone tissue comprisesa cancellous bone, a cortical bone, or a combination thereof. Accordingto another embodiment, the bone tissue comprises a cancellous bone.According to another embodiment, the cancellous bone is selected fromthe group consisting of a calcaneus, a distal femur bone, a proximalfemur, a proximal humerus, an ilium, a patella, a distal tibia, aproximal tibia, a scapula, a cancellous bone from a sternum, a talus, atleast one vertebral body, and a combination thereof. According toanother embodiment, the bone tissue comprises a cortical bone. Accordingto another embodiment, the bone tissue comprises periosteal tissue.According to another embodiment, the at least one viable population oftissuegenic cells is derived from a tissue selected from the groupconsisting of an adipose tissue, an amnion tissue, an artery tissue, abone tissue, a cartilage tissue, a chorion tissue, a colon tissue, adental tissue, a dermal tissue, a duodenal tissue, an endothelialtissue, an epithelial tissue, a fascia, a gastrointestinal tissue, agrowth plate tissue, an intervertebral disc tissue, an intestinalmucosal tissue, an intestinal serosal tissue, a ligament tissue, a livertissue, a lung tissue, a mammary tissue, a meniscal tissue, a muscletissue, a nerve tissue, an ovarian tissue, a parenchymal organ tissue, apericardial tissue, a periosteal tissue, a peritoneal tissue, aplacental tissue, a skin tissue, a spleen tissue, a stomach tissue, asynovial tissue, a tendon tissue, a testes tissue, an umbilical cordtissue, a urological tissue, a vascular tissue, a vein tissue, and acombination thereof. According to another embodiment, the at least oneviable population of tissuegenic cells adherent to and resident in anendogenous milieu of the growth conductive matrix comprises at least oneviable population selected from the group consisting of a viable stemcell population and a viable progenitor cell population. According toanother embodiment, the at least one viable population of tissuegeniccells comprises a population of viable multipotent stem cells. Accordingto another embodiment, the at least one viable population of tissuegeniccells comprises a population of viable pluripotent stem cells. Accordingto another embodiment, the at least one viable population of tissuegeniccells comprises a population of viable bone-derived osteoprogenitorcells. According to another embodiment, the at least one tissue-derivedgrowth-conductive matrix is derived from an autologous tissue. Accordingto another embodiment, the at least one growth-conductive matrix isderived from an allogeneic tissue. According to another embodiment, theendogenous niche of the growth conductive matrix comprises a tissuegeniccell niche.

According to another embodiment, the implant is an implantablecomposition comprising a growth-conductive matrix (e.g., anosteoconductive matrix including bone), a viable population oftissuegenic cells (e.g., osteogenic cells), and a growth-inductivematrix (e.g., demineralized bone matrix). In an embodiment, a secondgrowth-inductive matrix (e.g., a second demineralized bone matrix) isprovided separately from the aforesaid implantable composition foraddition to the implantable composition at a later time.

According to another aspect, the described invention provides a methodof fabricating a tissue-derived implant, the method comprising steps:(a) isolating a tissue comprising at least tissue-derived onegrowth-conductive matrix from which unwanted cells have been removed,wherein the growth-conductive matrix comprises at least one viablenonexpanded population of endogenous tissuegenic cells, and wherein thetissuegenic cells are adherent to and resident in an endogenous milieuof the growth-conductive matrix; (b) separating the at least onegrowth-conductive matrix from the tissue of (a) to generate a pluralityof separated matrix pieces comprising the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix, wherein the tissuegenic cells in theseparated matrix pieces is of a relative frequency substantially similarto that found in the growth matrix of step (a); (c) rinsing theplurality of the separated matrix pieces of (b) to form a plurality ofrinsed separated matrix pieces comprising the at least one viablepopulation of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix of (b), wherein thetissuegenic cells in the rinsed separated matrix pieces of step (c) isof a relative frequency substantially similar to that found in thegrowth-conductive matrix of step (a); (d) collecting the plurality ofthe rinsed separated matrix pieces of (c) comprising the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix of (c), wherein the atleast one viable tissuegenic cell population in the collected rinsedseparated matrix pieces is of a relative frequency substantially similarto that found in the growth-conductive matrix of step (a); (e) packagingthe plurality of the collected rinsed separated matrix pieces of (d)comprising at least one viable population of tissuegenic cells adherentto and resident in the endogenous milieu of the growth-conductivematrix, wherein the at least one viable tissuegenic cell population isof a relative frequency substantially similar to that found in thegrowth-conductive matrix of step (a), to form the implant.

According to one embodiment of the method, steps (a)-(d) are carried outat a temperature of about 4° C. to about 10° C. According to anotherembodiment, the at least one tissue-derived growth-conductive matrix isderived from a tissue selected from the group consisting of an adiposetissue, an amnion tissue, an artery tissue, a bone tissue, a cartilagetissue, a chorion tissue, a colon tissue, a dental tissue, a dermaltissue, a duodenal tissue, an endothelial tissue, an epithelial tissue,a fascial tissue, a gastrointestinal tissue, a growth plate tissue, anintervertebral disc tissue, an intestinal mucosal tissue, an intestinalserosal tissue, a ligament tissue, a liver tissue, a lung tissue, amammary tissue, a meniscal tissue, a muscle tissue, a nerve tissue, anovarian tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a placental tissue, a skintissue, a spleen tissue, a stomach tissue, a synovial tissue, a tendontissue, a testes tissue, an umbilical cord tissue, a urological tissue,a vascular tissue, a vein tissue, and a combination thereof. Accordingto another embodiment, the growth-conductive matrix and the tissuegeniccells are derived from a bone tissue. According to another embodiment,the bone tissue comprises a cancellous bone, a cortical bone, or acombination thereof. According to another embodiment, the bone tissuecomprises a cancellous bone. According to another embodiment, thecancellous bone is selected from the group consisting of a calcaneus, adistal femur bone, a proximal femur, a proximal humerus, an ilium, apatella, a distal tibia, a proximal tibia, a scapula, a cancellous bonefrom a sternum, a talus, at least one vertebral body, and a combinationthereof. According to another embodiment, the bone tissue comprises acortical bone. According to another embodiment, the bone tissuecomprises periosteal tissue. According to another embodiment, the methodfurther comprises step (f) supplementing the growth-conductive matrix ofstep (a) with at least one growth-inductive component. According toanother embodiment, the growth-inductive component is tissue-derived.According to another embodiment, the growth-inductive componentcomprises demineralized bone matrix, which may be derived from corticalbone. According to another embodiment, the growth-inductive componentoriginates from a component of the growth-inductive component other thancells. According to another embodiment, the growth-conductive componentcomprises a growth medium derived from expanded tissuegenic cells.According to another embodiment, the at least one growth-inductivecomponent comprises at least one cytokine According to anotherembodiment, the at least one growth-inductive component comprises atleast one growth factor. According to another embodiment, the least onegrowth factor is selected from the group consisting of fibroblast growthfactor-2 (FGF-2), fibroblast growth factor-5 (FGF-5), insulin-likegrowth factor 1 (IGF-1), neural epidermal growth-factor-like 1 (Nel-like1, NELL1), transforming growth factor beta (TGF-β), a bone morphogenicprotein, bone morphogenic protein-2 (BMP-2), bone morphogenic protein-7(BMP-7), platelet-derived growth factor (PDGF), vascular endothelialgrowth factor (VEGF), and a combination thereof. According to anotherembodiment, the tissue-derived growth-conductive matrix is derived froma cadaveric donor. According to another embodiment, the tissue-derivedgrowth conductive matrix is derived from a living donor. According toanother embodiment, the tissue-derived growth-conductive matrix isderived from an autologous tissue. According to another embodiment, theat least one growth-conductive matrix is derived from an allogeneictissue. According to another embodiment, the endogenous niche of thegrowth conductive matrix comprises a tissuegenic cell niche. Accordingto another embodiment, the at least one viable population of tissuegeniccells adherent to and resident in an endogenous milieu of the growthconductive matrix comprises at least one viable population selected fromthe group consisting of a viable stem cell population and a viableprogenitor cell population. According to another embodiment, the atleast one viable population of tissuegenic cells comprises at least oneviable pluripotent stem cell population. According to anotherembodiment, the at least one viable population of tissuegenic cellscomprises at least one viable multipotent stem cell population.According to another embodiment, the at least one viable population oftissuegenic cells is derived from a tissue selected from the groupconsisting of an adipose tissue, an amnion tissue, an artery tissue, abone tissue, a cartilage tissue, a chorion tissue, a colon tissue, adental tissue, a dermal tissue, a duodenal tissue, an endothelialtissue, an epithelial tissue, a fascial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a ligamenttissue, a liver tissue, a lung tissue, a mammary tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, aparenchymal organ tissue, a pericardial tissue, a periosteal tissue, aperitoneal tissue, a placental tissue, a skin tissue, a spleen tissue, astomach tissue, a synovial tissue, a tendon tissue, a testes tissue, anumbilical cord tissue, a urological tissue, a vascular tissue, a veintissue, and a combination thereof. According to another embodiment,separating step (b) comprises mincing the tissue to yield a plurality ofgrowth-conductive matrix pieces. According to another embodiment,separating step (b) comprises cutting the tissue to yield a plurality ofgrowth-conductive matrix pieces. According to another embodiment,separating step (b) comprises milling the tissue to yield a plurality ofgrowth-conductive matrix pieces. According to another embodiment,rinsing step (c) comprises admixing the plurality of separatedgrowth-conductive matrix pieces of (b) comprising the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix with a liquid.According to another embodiment, the plurality of the separated matrixpieces of (b) is rinsed with a buffered isotonic solution.

According to another aspect, the described invention provides a methodof treating a bony defect at a defect site in a subject (e.g., apatient) in need thereof, comprising steps: (a) providing atissue-derived orthopedic implant comprising (i) a plurality of piecescomprising at least one tissue-derived growth conductive matrix; and(ii) at least one viable population of tissuegenic cells adherent to andresident in an endogenous milieu of the growth conductive matrix,wherein the at least one population of tissuegenic cells is present inthe growth conductive matrix at a relative frequency substantiallysimilar to the relative frequency of the tissuegenic cells found invivo; (b) implanting the orthopedic implant at the defect site; and (c)filling the bony defect.

According to one embodiment of the method, the bony defect resulted fromtumor surgery. According to another embodiment, the bony defect resultedfrom a traumatic injury. According to another embodiment, the bonydefect resulted from a congenital skeletal abnormality. According toanother embodiment, the bony defect resulted from a fracture. Accordingto another embodiment, the bony defect resulted from a spinalarthrodesis. According to another embodiment, the bony defect resultedfrom scoliosis. According to another embodiment, the tissue-derivedgrowth conductive matrix is derived from a mammalian tissue. Accordingto another embodiment, the at least one tissue-derived growth conductivematrix is derived from a tissue selected from the group consisting of anadipose tissue, an amnion tissue, an artery tissue, a bone tissue, acartilage tissue, a chorion tissue, a colon tissue, a dental tissue, adermal tissue, a duodenal tissue, an endothelial tissue, an epithelialtissue, a fascial tissue, a gastrointestinal tissue, a growth platetissue, an intervertebral disc tissue, an intestinal mucosal tissue, anintestinal serosal tissue, a ligament tissue, a liver tissue, a lungtissue, a mammary tissue, a meniscal tissue, a muscle tissue, a nervetissue, an ovarian tissue, a parenchymal organ tissue, a pericardialtissue, a periosteal tissue, a peritoneal tissue, a placental tissue, askin tissue, a spleen tissue, a stomach tissue, a synovial tissue, atendon tissue, a testes tissue, an umbilical cord tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.According to another embodiment, the tissue-derived growth conductivematrix is derived from a human donor. According to another embodiment,the tissue-derived growth conductive matrix is derived from a cadavericdonor. According to another embodiment, the tissue-derived growthconductive matrix is derived from a living donor. According to anotherembodiment, the at least one tissue-derived growth conductive matrix isderived from an autologous tissue. According to another embodiment, theat least one growth conductive matrix is derived from an allogeneictissue. According to another embodiment, the endogenous milieu of thegrowth conductive matrix comprises a tissuegenic cell niche. Accordingto another embodiment, the at least one viable population of tissuegeniccells adherent to and resident in an endogenous milieu of the growthconductive matrix comprises at least one viable population selected fromthe group consisting of a viable stem cell population and a viableprogenitor cell population. According to another embodiment, the atleast one viable population of tissuegenic cells comprises at least oneviable pluripotent stem cell population. According to anotherembodiment, the viable population of tissuegenic cells comprises atleast one viable multipotent stem cell population. According to anotherembodiment, the at least one viable population of tissuegenic cellscomprises a population of viable bone-derived osteoprogenitor cells.According to another embodiment, the at least one viable population oftissuegenic cells is derived from a tissue selected from the groupconsisting of an adipose tissue, an amnion tissue, a bone tissue, acartilage tissue, a chorion tissue, a dental tissue, a dermal tissue, agastrointestinal tissue, an intervertebral disc tissue, an epithelialtissue, a fascial tissue, a growth plate tissue, a ligament tissue, alung tissue, a liver tissue, a mammary tissue, a meniscal tissue, amuscle tissue, a nerve tissue, a periosteal tissue, a placental tissue,a skin tissue, a synovial tissue, a tendon tissue, an umbilical cordtissue, a urological tissue, a vascular tissue, and a combinationthereof. According to another embodiment, the implant further comprisesat least one growth-inductive component. According to anotherembodiment, the growth-inductive component is tissue-derived. Accordingto another embodiment, the growth-inductive component comprisesdemineralized bone matrix, which may be derived from cortical bone.According to another embodiment, the tissue-derived growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to anotherembodiment, the growth-conductive component comprises a growth mediumderived from expanded tissuegenic cells. According to anotherembodiment, the tissuegenic cells adherent to and resident in theendogenous milieu of the growth conductive matrix secrete the at leastone growth-inductive component. According to another embodiment, the atleast one growth-inductive component comprises at least one cytokineAccording to another embodiment, the at least one growth-inductivefactor comprises at least one growth factor. According to anotherembodiment, the at least one growth factor is selected from the groupconsisting of fibroblast growth factor-2 (FGF-2), fibroblast growthfactor-5 (FGF-5), insulin-like growth factor 1 (IGF-1), neural epidermalgrowth-factor-like 1 (Nel-like 1, NELL1), transforming growth factorbeta (TGF-β), a bone morphogenic protein, bone morphogenic protein-2(BMP-2), bone morphogenic protein-7 (BMP-7), platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF), and acombination thereof. According to another embodiment, the growthconductive matrix and the tissuegenic cells are derived from a bonetissue. According to another embodiment, the bone tissue comprises ascancellous bone, a cortical bone, or a combination thereof. According toanother embodiment, the bone tissue comprises a cancellous bone.According to another embodiment, the cancellous bone is selected fromthe group consisting of a calcaneus, a distal femur bone, a proximalfemur, a proximal humerus, an ilium, a patella, a distal tibia, aproximal tibia, a scapula, a cancellous bone from a sternum, a talus, atleast one vertebral body, and a combination thereof. According toanother embodiment, the bone tissue comprises a cortical bone. Accordingto another embodiment, the bone tissue comprises periosteal tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference ismade to the following detailed description of exemplary embodimentsconsidered in conjunction with the accompanying drawings, in which:

FIG. 1 shows a plot of BMP-2 (pg/g DCB) versus time (weeks). The levelsof BMP-2 remain about or above 10,000 pg/g DCB after 12 weeks post-thaw.

FIG. 2 shows a plot of the MLR response of a positive control (latexglove), a negative control, DCB, and 3 sample orthopedic implants.

FIG. 3A shows a plot of the complement activation response of c3aprotein (ng/ml) of a positive control, negative control, untreatedplasma, and 4 sample orthopedic implants. FIG. 3B shows a plot of thecomplement activation response of SC5b (ng/ml) protein of a positivecontrol, negative control, untreated plasma, and 4 sample orthopedicimplants.

FIG. 4A shows that at day 14 hypotrophic chondrocytes are beginning toform; FIG. 4B shows that at day 28, new bone formation is evident.

DETAILED DESCRIPTION Glossary

The term “ambient temperature” as used herein refers to the temperatureof the immediate, unaltered surroundings. Ambient temperature is betweenabout 18° C. and about 28° C. According to some embodiments, ambienttemperature is room temperature.

The term “adherent” as used herein refers to the act of sticking to,clinging, or staying attached.

The term “adipokine” as used herein refers to a factor secreted byadipose tissue.

The term “adipocyte” as used herein refers to the functional cell typeof fat, or adipose tissue, that is found throughout the body,particularly under the skin. Adipocytes store and synthesize fat forenergy, thermal regulation and cushioning against mechanical shock.Although the lineage of adipocytes is still unclear, it appears thatMSCs can differentiate into two types of lipoblasts, one that give riseto white adipocytes and the other to brown adipocytes. Both types ofadipocytes store fat.

The term “adipogenic” as used herein refers to a potential ofundifferentiated precursor cells to differentiate into fat forming oradipocompetent cells.

The term “adipose stem cell” (ASC) as used herein refers to pluripotentstem cells, MSCs and more committed adipose progenitors and stromaobtained from adipose tissue.

The term “administer” as used herein means to give or to apply.

The term “allogeneic” as used herein refers to being geneticallydifferent although belonging to or obtained from the same species.

The term “amniotic stem cells” as used herein refers to pluripotent stemcells, multipotent stem cells and progenitor cells derived from amnioticmembrane, which can give rise to a limited number of cell types in vitroand/or in vivo under an appropriate condition, and expressly includesboth amniotic epithelial cells and amniotic stromal cells.

The term “attached” as used herein refers to being fastened, fixed,joined, connected, bound, adhered to or assembled with.

The term “autologous” as used herein means derived from the sameorganism.

The term “autologous graft” or “autograft” as used herein refers to atissue that is grafted into a new position in or on the body of the sameindividual.

The term “basic fibroblast growth factor” (bFGF) as used herein refersto a multifunctional effector for many cells of mesenchymal andneuroectodermal origin that is a potent inducer of neovascularizationand angiogenesis.

The term “biocompatible” as used herein refers to causing no clinicallyrelevant tissue irritation, injury, toxic reaction, or immunologicalreaction to living tissue.

The term “biomarkers” (or “biosignatures”) as used herein refers topeptides, proteins, nucleic acids, antibodies, genes, metabolites, orany other substances used as indicators of a biologic state. It is acharacteristic that is measured objectively and evaluated as a cellularor molecular indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention.

The term “bone” as used herein refers to a hard connective tissueconsisting of cells embedded in a matrix of mineralized ground substanceand collagen fibers. The fibers are impregnated with a form of calciumphosphate similar to hydroxyapatite as well as with substantialquantities of carbonate, citrate and magnesium. Bone consists of a denseouter layer of compact substance or cortical substance covered by theperiosteum and an inner loose, spongy substance; the central portion ofa long bone is filled with marrow.

The terms “cancellous bone” or “trabecular bone” as used herein refer tothe spongy bone found in the inner parts of compact bone in which thematrix forms a lattice of large plates and rods known as the trabeculae,which anastomose to form a latticework. This latticework partiallyencloses many intercommunicating spaces filled with bone marrow. Themarrow spaces are relatively large and irregularly arranged, and thebone substance is in the form of slender anastomosing trabeculae andpointed spicules.

The terms “cortical bone” or “compact bone” as used herein refer to thedense outer layer of bone that consists largely of concentric lamellarosteons and interstitial lamellae. The spaces or channels are narrow andthe bone substance is densely packed.

The term “bone morphogenetic protein (BMP) as used herein refers to agroup of cytokines that are part of the transforming growth factor-β(TGF-β) superfamily. BMP ligands bind to a complex of the BMP receptortype II and a BMP receptor type I (Ia or Ib). This leads to thephosphorylation of the type I receptor that subsequently phosphorylatesthe BMP-specific Smads (Smad1, Smad5, and Smad8), allowing thesereceptor-associated Smads to form a complex with Smad4 and move into thenucleus where the Smad complex binds a DNA binding protein and acts as atranscriptional enhancer. BMPs have a significant role in bone andcartilage formation in vivo. It has been reported that most BMPs areable to stimulate osteogenesis in mature osteoblasts, while BMP-2, 6,and 9 may play an important role in inducing osteoblast differentiationof mesenchymal stem cells. Cheng, H. et al., J. Bone & Joint Surgery 85:1544-52 (2003).

The term “bound” or any of its grammatical forms as used herein refersto the capacity to hold onto, attract, interact with or combine with.

The term “buffer” or “buffer solution” as used herein refers to acompound, usually a salt, which, when dissolved in an aqueous medium,serves to maintain the free hydrogen ion concentration of the solutionwithin a certain pH range when hydrogen ions are added or removed fromthe solution. A salt or solution is said to have a “buffering capacity”or to buffer the solution over such a range, when it provides thisfunction. Generally a buffer will have adequate buffering capacity overa range that is within .±. 1 pH unit of its pK.

The term “buffered isotonic solution” as used herein refers to anybuffer that is commonly used in biological research. Exemplary bufferedisotonic solutions include but are not limited to balanced salt solution(BSS), Hank's Balanced Salt Solution, Gey's Balanced Salt Solution,Hank's Buffered Salt Solution, Phosphate Buffered Saline, Tris-BufferedSaline, etc. The term “isotonic solution” as used herein refers to asolution whose osmolarity and ion concentrations closely match thosewithin normal cells of the body and the blood.

The term “carrier” as used herein refer to a pharmaceutically acceptableinert agent or vehicle for delivering one or more active agents to asubject, and often is referred to as “excipient.” The carrier must be ofsufficiently high purity and of sufficiently low toxicity to render itsuitable for administration to the subject being treated. The carrierfurther should maintain the stability and bioavailability of an activeagent

Stem Cell Markers

Coating the surface of every cell in the body are specialized proteins(“receptors”) capable of selectively binding or adhering to other“signaling” molecules. Normally, cells use these receptors and themolecules that bind to them as a way of communicating with other cellsand to carry out their proper functions in the body. These cell surfacereceptors are the stem cell markers. Each cell type has a certaincombination of receptors on their surface that makes themdistinguishable from other kinds of cells.

The cluster of differentiation (CD) system is a protocol used for theidentification of cell surface molecules. CD molecules can act innumerous ways, often acting as receptors or ligands; by which a signalcascade is initiated, altering the behavior of the cell. Some CDproteins do not play a role in cell signaling, but have other functions,such as cell adhesion. Generally, a proposed surface molecule isassigned a CD number once two specific monoclonal antibodies (mAb) areshown to bind to the molecule. If the molecule has not beenwell-characterized, or has only one mAb, the molecule usually is giventhe provisional indicator “w.”

The CD system nomenclature commonly used to identify cell markers thusallows cells to be defined based on what molecules are present on theirsurface. These markers often are used to associate cells with certainfunctions. While using one CD molecule to define populations isuncommon, combining markers has allowed for cell types with veryspecific definitions. More than 350 CD molecules have been identifiedfor humans.

CD molecules are utilized in cell sorting using various methods,including flow cytometry. Cell populations usually are defined using a“+” or a “−” symbol to indicate whether a certain cell fractionexpresses or lacks a particular CD molecule.

Table 8 identifies markers commonly used to identify stem cells and tocharacterize differentiated cell types:

TABLE 8 Commonly-Used Stem Cell Surface Surface Markers andCorresponding Differentiated Cell Types Marker Name Cell TypeSignificance Blood Vessel Fetal liver kinase-1 Endothelial Cell-surfacereceptor protein that identifies (Flkl) endothelial cell progenitor;marker of cell-cell contacts Smooth muscle Smooth muscle Identifiessmooth muscle cells in the wall of blood cell-specific vessels myosinheavy chain Vascular Smooth muscle Identifies smooth muscle cells in thewall of blood endothelial cell vessels cadherin Bone Bone-specificOsteoblast Enzyme expressed in osteoblast; activity indicates alkalinebone formation phosphatase (BAP) Hydroxyapatite Osteoblast Mineralizedbone matrix that provides structural integrity; marker of bone formationOsteocalcin (OC) Osteoblast Mineral-binding protein synthesized byosteoblast; marker of bone formation Bone Marrow and Blood BoneMesenchymal stem Important for the differentiation of committedmorphogenetic and progenitor cells mesenchymal cell types frommesenchymal stem protein receptor and progenitor cells; BMPR identifiesearly (BMPR) mesenchymal lineages (stem and progenitor cells) CD4 andCD8 White blood cell Cell-surface protein markers specific for mature T(WBC) lymphocyte (WBC subtype) CD34 Hematopoietic stem Cell-surfaceprotein on bone marrow cell, cell (HSC), satellite, indicative of a HSCand endothelial progenitor; endothelial progenitor CD34 also identifiesmuscle satellite, a muscle stem cell CD34⁺Sca1⁺ Lin⁻ Mesenchymal stemIdentifies MSCs, which can differentiate into profile cell (MSC)adipocyte, osteocyte, chondrocyte, and myocyte CD38 Absent on HSCCell-surface molecule that identifies WBC Present on WBC lineages.Selection of CD34⁺/VCD38⁻ cells allows lineages for purification of HSCpopulations CD44 Mesenchymal A type of cell-adhesion molecule used toidentify specific types of mesenchymal cells c-Kit HSC, MSC Cell-surfacereceptor on BM cell types that identifies HSC and MSC; binding by fetalcalf serum (FCS) enhances proliferation of ES cells, HSCs, MSCs, andhematopoietic progenitor cells Colony-forming HSC, MSC progenitor CFUassay detects the ability of a single stem cell unit (CFU) or progenitorcell to give rise to one or more cell lineages, such as red blood cell(RBC) and/or white blood cell (WBC) lineages Fibroblast colony- Bonemarrow An individual bone marrow cell that has given rise forming unit(CFU- fibroblast to a colony of multipotent fibroblastic cells; such F)identified cells are precursors of differentiated mesenchymal lineagesHoechst dye Absent on HSC Fluorescent dye that binds DNA; HSC extrudesthe dye and stains lightly compared with other cell types Leukocytecommon WBC Cell-surface protein on WBC progenitor antigen (CD45) Lineagesurface HSC, MSC Thirteen to 14 different cell-surface proteins thatantigen (Lin) Differentiated RBC are markers of mature blood celllineages; and WBC lineages detection of Lin-negative cells assists inthe purification of HSC and hematopoietic progenitor populations Mac-1WBC Cell-surface protein specific for mature granulocyte and macrophage(WBC subtypes) Muc-18 (CD146) Bone marrow Cell-surface protein(immunoglobulin superfamily) fibroblasts, found on bone marrowfibroblasts, which may be endothelial important in hematopoiesis; asubpopulation of Muc-18+ cells are mesenchymal precursors Stem cellantigen HSC, MSC Cell-surface protein on bone marrow (BM) cell, (Sca-1)indicative of HSC and MSC Bone Marrow and Blood cont. Stro-1 antigenStromal Cell-surface glycoprotein on subsets of bone (mesenchymal)marrow stromal (mesenchymal) cells; selection of precursor cells,Stro-1+ cells assists in isolating mesenchymal hematopoietic cellsprecursor cells, which are multipotent cells that give rise toadipocytes, osteocytes, smooth myocytes, fibroblasts, chondrocytes, andblood cells Thy-1 HSC, MSC Cell-surface protein; negative or lowdetection is suggestive of HSC Cartilage Collagen types H ChondrocyteStructural proteins produced specifically by and IV chondrocyte KeratinKeratinocyte Principal protein of skin; identifies differentiatedkeratinocyte Sulfated Chondrocyte Molecule found in connective tissues;synthesized proteoglycan by chondrocyte Fat Adipocyte lipid- AdipocyteLipid-binding protein located specifically in binding protein adipocyte(ALBP) Fatty acid Adipocyte Transport molecule located specifically intransporter (FAT) adipocyte Adipocyte lipid- Adipocyte Lipid-bindingprotein located specifically in binding protein adipocyte (ALBP) LiverAlbumin Hepatocyte Principal protein produced by the liver; indicatesfunctioning of maturing and fully differentiated hepatocytes B-1integrin Hepatocyte Cell-adhesion molecule important in cell-cellinteractions; marker expressed during development of liver NervousSystem CD133 Neural stem cell, HSC Cell-surface protein that identifiesneural stem cells, which give rise to neurons and glial cells Glialfibrillary Astrocyte Protein specifically produced by astrocyte acidicprotein (GFAP) Microtubule- Neuron Dendrite-specific MAP; protein foundspecifically associated protein- in dendritic branching of neuron 2(MAP-2) Myelin basic Oligodendrocyte Protein produced by matureoligodendrocytes; protein (MPB) located in the myelin sheath surroundingneuronal structures Nestin Neural progenitor Intermediate filamentstructural protein expressed in primitive neural tissue Neural tubulinNeuron Important structural protein for neuron; identifiesdifferentiated neuron Neurofilament Neuron Important structural proteinfor neuron; identifies (NF) differentiated neuron Neurosphere Embryoidbody (EB), Cluster of primitive neural cells in culture of ESdifferentiating ES cells; indicates presence of early neurons and gliaNoggin Neuron A neuron-specific gene expressed during the development ofneurons O4 Oligodendrocyte Cell-surface marker on immature, developingoligodendrocyte O1 Oligodendrocyte Cell-surface marker thatcharacterizes mature oligodendrocyte Synaptophysin Neuron Neuronalprotein located in synapses; indicates connections between neurons TauNeuron Type of MAP; helps maintain structure of the axon PancreasCytokeratin 19 Pancreatic epithelium CK19 identifies specific pancreaticepithelial cells (CK19) that are progenitors for islet cells and ductalcells Glucagon Pancreatic islet Expressed by alpha-islet cell ofpancreas Insulin Pancreatic islet Expressed by beta-islet cell ofpancreas Insulin-promoting Pancreatic islet Transcription factorexpressed by beta-islet cell of factor-1 (PDX-1) pancreas NestinPancreatic progenitor Structural filament protein indicative ofprogenitor cell lines including pancreatic Pancreatic Pancreatic isletExpressed by gamma-islet cell of pancreas polypeptide SomatostatinPancreatic islet Expressed by delta-islet cell of pancreas PluripotentStem Cells Alkaline Embryonic stem (ES), Elevated expression of thisenzyme is associated phosphatase embryonal carcinoma withundifferentiated pluripotent stem cell (PSC) (EC) Alpha-fetoproteinEndoderm Protein expressed during development of primitive (AFP)endoderm; reflects endodermal differentiation Pluripotent Stem CellsBone Mesoderm Growth and differentiation factor expressed duringmorphogenetic early mesoderm formation and differentiation protein-4Brachyury Mesoderm Transcription factor important in the earliest phasesof mesoderm formation and differentiation; used as the earliestindicator of mesoderm formation Cluster designation ES, EC Surfacereceptor molecule found specifically on 30 (CD30) PSC Cripto (TDGF-1)ES, cardiomyocyte Gene for growth factor expressed by ES cells,primitive ectoderm, and developing cardiomyocyte GATA-4 gene EndodermExpression increases as ES differentiates into endoderm GCTM-2 ES, ECAntibody to a specific extracellular-matrix molecule that is synthesizedby undifferentiated PSCs Genesis ES, EC Transcription factor uniquelyexpressed by ES cells either in or during the undifferentiated state ofPSCs Germ cell nuclear ES, EC Transcription factor expressed by PSCsfactor Hepatocyte nuclear Endoderm Transcription factor expressed earlyin endoderm factor-4 (HNF-4) formation Nestin Ectoderm, neural andIntermediate filaments within cells; characteristic pancreaticprogenitor of primitive neuroectoderm formation Neuronal cell- EctodermCell-surface molecule that promotes cell-cell adhesion moleculeinteraction; indicates primitive neuroectoderm (N-CAM) formationOCT4/POU5F1 ES, EC Transcription factor unique to PSCs; essential forestablishment and maintenance of undifferentiated PSCs Pax6 EctodermTranscription factor expressed as ES cell differentiates intoneuroepithelium Stage-specific ES, EC Glycoprotein specificallyexpressed in early embryonic antigen- embryonic development and byundifferentiated 3 (SSEA-3) PSCs Stage-specific ES, EC Glycoproteinspecifically expressed in early embryonic antigen- embryonic developmentand by undifferentiated 4 (SSEA-4) PSCs Stem cell factor ES, EC, HSC,MSC Membrane protein that enhances proliferation of (SCF or c-Kit ES andEC cells, hematopoietic stem cell (HSCs), ligand) and mesenchymal stemcells (MSCs); binds the receptor c-Kit Telomerase ES, EC An enzymeuniquely associated with immortal cell lines; useful for identifyingundifferentiated PSCs TRA-1-60 ES, EC Antibody to a specificextracellular matrix molecule is synthesized by undifferentiated PSCsTRA-1-81 ES, EC Antibody to a specific extracellular matrix moleculenormally synthesized by undifferentiated PSCs Vimentin Ectoderm, neuraland Intermediate filaments within cells; characteristic pancreaticprogenitor of primitive neuroectoderm formation SkeletalMuscle/Cardiac/Smooth Muscle MyoD and Pax7 Myoblast, myocyteTranscription factors that direct differentiation of myoblasts intomature myocytes Myogenin and Skeletal myocyte Secondary transcriptionfactors required for MR4 differentiation of myoblasts from muscle stemcells Myosin heavy Cardiomyocyte A component of structural andcontractile protein chain found in cardiomyocyte Myosin light chainSkeletal myocyte A component of structural and contractile protein foundin skeletal myocyte

Table 9 shows commonly used markers employed by skilled artisans toidentify and characterize differentiated white blood cell types:

TABLE 9 List of Surface Markers on White Blood Cell Types Type of CellCD Markers Stem cells CD34+, CD31− All leukocyte groups CD45+Granulocyte CD45+, CD15+ Monocyte CD45+, CD14+ T lymphocyte CD45+, CD3+T helper cell CD45+, CD3+, CD4+ Cytotoxic T cell CD45+, CD3+, CD8+ Blymphocyte CD45+, CD19+ or CD45+, CD20+ Thrombocyte CD45+, CD61+ Naturalkiller cell CD16+, CD56+, CD3−

Table 10 correlates the exemplary protein expression profile of adiposederived stem cells (ASCs) with the corresponding surface markers (Flynnet. al., 2208 Organogenesis, 4(4): 228-235; Gronthos et. al., 2011, J.Cell. Physiol., 189: 54-63).

TABLE 10 Adipose-derived Stem Cell Protein Expression and Surface MarkerProfile Class Protein Marker Cell Adhesion Integrin β₁ CD29 Integrin α₄CD49_(d) Integrin a_(a) CD49_(e) Vascular Cell VCAM; CD106 AdhesionMolecule Intracellular Adhesion ICAM; CD54 Molecule −1 ActivatedLeukocyte Cell ALCAM; CD166 Adhesion Molecule Tetraspan CD9 EndoglinCD105 Muc18 CD146 Receptors Hyaluronate receptor CD44 Transferrinreceptor CD71 Insulin receptor Glucocorticoid receptor Triiodothyronine(T3) receptor Retinoic acid receptor ECM Collagen type I Collagen typeIII Collagen type IV Collagen type VI CD68 Osteopontin OsteonectinCytoskeletal A-smooth muscle actin Vimentin Other HLA-ABC Majorhistocompatibility complex class I antigen DAF CD55 Complement protectinCD59

CD3 (TCR complex) is a protein complex composed of four distinct chains.In mammals, the complex contains a CD3γ chain, a CD3δ chain, and twoCD3ε chains, which associate with the T cell receptor (TCR) and theζ-chain to generate an activation signal in T lymphocytes. Together, theTCR, the ζ-chain and CD3 molecules comprise the TCR complex. Theintracellular tails of CD3 molecules contain a conserved motif known asthe immunoreceptor tyrosine-based activation motif (ITAM), which isessential for the signaling capacity of the TCR. Upon phosphorylation ofthe ITAM, the CD3 chain can bind ZAP70 (zeta associated protein), akinase involved in the signaling cascade of the T cell.

Integrins are receptors that mediate attachment between a cell and thetissues surrounding it and are involved in cell-cell and cell-matrixinteractions. In mammals, 18 α and 8 β subunits have been characterized.Both α and β subunits contain two separate tails, both of whichpenetrate the plasma membrane and possess small cytoplasmic domains.

Integrin αM (ITGAM; CD11b; macrophage-1 antigen (Mac-1); complementreceptor 3 (CR3)) is a protein subunit of the heterodimeric integrinαMβ2 molecule. The second chain of αMβ2 is the common integrin β2subunit (CD18). αMβ2 is expressed on the surface of many leukocytesincluding monocytes, granulocytes, macrophages and natural killer cells.It generally is believed that αMβ2 mediates inflammation by regulatingleukocyte adhesion and migration. Further, αMβ2 is thought to have arole in phagocytosis, cell-mediated cytotoxicity, chemotaxis andcellular activation, as well as being involved in the complement systemdue to its capacity to bind inactivated complement component 3b (iC3b).The ITGAM subunit of integrin αMβ2 is involved directly in causing theadhesion and spreading of cells, but cannot mediate cellular migrationwithout the presence of the β2 (CD18) subunit.

CD14 is a cell surface protein expressed mainly by macrophages and, to alesser extent, neutrophil granulocytes. CD14+ cells are monocytes thatcan differentiate into a host of different cells; for example,differentiation to dendritic cells is promoted by cytokines such asGM-CSF and IL-4. CD14 acts as a co-receptor (along with toll-likereceptor (TLR) 4 and lymphocyte antigen 96 (MD-2)) for the detection ofbacterial lipopolysaccharide (LPS). CD14 only can bind LPS in thepresence of lipopolysaccharide binding protein (LBP).

CD15 (3-fucosyl-N-acetyl-lactosamine; stage specific embryonic antigen 1(SSEA-1)) is a carbohydrate adhesion molecule that can be expressed onglycoproteins, glycolipids and proteoglycans. CD15 commonly is found onneutrophils and mediates phagocytosis and chemotaxis.

CD16 is an Fc receptor (FcγRIIIa and FcγRIIIb) found on the surface ofnatural killer cells, neutrophil polymorphonuclear leukocytes, monocytesand macrophages. Fc receptors bind to the Fc portion of IgG antibodies.

CD19 is a human protein expressed on follicular dendritic cells and Bcells. This cell surface molecule assembles with the antigen receptor ofB lymphocytes in order to decrease the threshold for antigenreceptor-dependent stimulation. It generally is believed that, uponactivation, the cytoplasmic tail of CD19 becomes phosphorylated, whichallows binding by Src-family kinases and recruitment of phosphoinositide3 (PI-3) kinases.

CD20 is a non-glycosylated phosphoprotein expressed on the surface ofall mature B-cells. Studies suggest that CD20 plays a role in thedevelopment and differentiation of B-cells into plasma cells. CD20 isencoded by a member of the membrane-spanning 4A gene family (MS4A).Members of this protein family are characterized by common structuralfeatures and display unique expression patterns among hematopoieticcells and nonlymphoid tissues.

CD31 (platelet/endothelial cell adhesion molecule; PECAM1) normally isfound on endothelial cells, platelets, macrophages and Kupffer cells,granulocytes, T cells, natural killer cells, lymphocytes,megakaryocytes, osteoclasts and neutrophils. CD31 has a key role intissue regeneration and in safely removing neutrophils from the body.Upon contact, the CD31 molecules of macrophages and neutrophils are usedto communicate the health status of the neutrophil to the macrophage.

CD34 is a monomeric cell surface glycoprotein normally found onhematopoietic cells, endothelial progenitor cells, endothelial cells ofblood vessels, and mast cells. The CD34 protein is a member of a familyof single-pass transmembrane sialomucin proteins and functions as acell-cell adhesion factor. Studies suggest that CD34 also may mediatethe attachment of stem cells to bone marrow extracellular matrix ordirectly to stromal cells.

CD44 (the “hyaluronan receptor”), a cell-surface glycoprotein involvedin cell-cell interactions, cell adhesion and migration, is used toidentify specific types of mesenchymal cells.

CD45 (protein tyrosine phosphatase, receptor type, C; PTPRC) cellsurface molecule is expressed specifically in hematopoietic cells. CD45is a protein tyrosine phosphatase (PTP) with an extracellular domain, asingle transmembrane segment, and two tandem intracytoplasmic catalyticdomains, and thus belongs to receptor type PTP. Studies suggest it is anessential regulator of T-cell and B-cell antigen receptor signaling thatfunctions by direct interaction with components of the antigen receptorcomplexes, or by activating various Src family kinases required forantigen receptor signaling. CD45 also suppresses JAK kinases, and thusfunctions as a regulator of cytokine receptor signaling. The CD45 familyconsists of multiple members that are all products of a single complexgene. Various known isoforms of CD45 include: CD45RA, CD45RB, CD45RC,CD45RAB, CD45RAC, CD45RBC, CD45RO, and CD45R (ABC). Different isoformsmay be found on different cells. For example, CD45RA is found on naïve Tcells and CD45RO is found on memory T cells.

CD56 (neural cell adhesion molecule, NCAM) is a homophilic bindingglycoprotein expressed on the surface of neurons, glia, skeletal muscleand natural killer cells. It generally is believed that NCAM has a rolein cell-cell adhesion, neurite outgrowth, and synaptic plasticity. Thereare three known main isoforms of NCAM, each varying only in theircytoplasmic domains: NCAM-120 kDA (glycosylphopharidylinositol (GPI)anchored); NCAM-140 kDa (short cytoplasmic domain); and NCAM (longcytoplasmic domain). The different domains of NCAM have different roles,with the Ig domains being involved in homophilic binding to NCAM, andthe fibronectin type III (FNIII) domains being involved in signalingleading to neurite outgrowth.

CD59 refers to a glycosylphosphatidylinositol (GPI)-linked membraneglycoprotein which protects human cells from complement-mediated lysis.

The CD66 antigen family identifies a neutrophil-specific epitope withinthe hematopoietic system that is expressed by members of thecarcinoembryonic antigen family of adhesion molecules, which belongwithin the immunoglobulin gene superfamily. The extracellular portionsof all CD66 (a-f) molecules possess a N-terminal V-set IgSF domainwhich, lacks the canonical inter-b-sheet disulfide of the CD-2 family.CD66a is heavily glycosylated type 1 glycoprotein with more than 60% ofthe mass contributed by N-linked glycans, which bear sialylated Lex (sLex, CD15s) structures. In CD66a they are spaced further apart,VxYxxLx21IxYxxV, and resemble motifs which bind tyrosine phosphatasessuch as SHIP-1 and -2. Activation of neutrophils leads tophosphorylation of tyrosine residues in the CD66a cytoplasmic domain.CD66a is expressed on granulocytes and epithelial cells. Products of 4of the 7 functional carcinoembryonic antigen (CEA) family genes,CD66a-d, are known to be expressed on hematopoietic cells. Theexpression of these molecules on hematopoietic cells is generallyrestricted to the myeloid lineage. These molecules are present at lowlevels on resting mature granulocytes but expression increases rapidlyfollowing activation with inflammatory agonists, probably as a result ofexocytosis from storage granules. CD66a is detected on some macrophagesin tissue sections and has been reported on T cells and a subpopulationof activated NK cells.

CD66b ((CGM1); CD67, CGM6, NCA-95) is a glycosylphosphatidylinositol(GPI)-linked protein that is a member of the immunoglobulin superfamilyand carcinoembryonic antigen (CEA)-like subfamily. CD66b, expressed ongranulocytes, generally is believed to be involved in regulatingadhesion and activation of human eosinophils.

CD90 or Thy-1 is a 25-37 kDa heavily N-glycosylated,glycophosphatidylinositol (GPI) anchored conserved cell surface proteinwith a single V-like immunoglobulin domain, originally discovered as athymocyte antigen. It belongs to the immunoglobulin gene superfamily.The complex carbohydrate side chains vary in composition between tissuesand species. Generally, CD90 is expressed on hematopoietic stem cellsand neurons. CD90 is highly expressed in connective tissue, on variousfibroblast and stromal cell lines and is expressed on all thymocytes andperipheral T cells in mice. In humans, CD90 is expressed only on a smallnumber of fetal thymocytes, 10%-40% of blood CD34+ cells in bone marrow,and <1% of CD3+CD4+ lymphocytes in peripheral circulation. CD90 also isexpressed in the human lymph node HEV endothelium but not on otherendothelia and lastly, is expressed on a limited number oflymphoblastoid and leukemic cell lines.

CD105 (endoglin) is a homodimeric integral membrane glycoproteincomposed of disulfide-linked subunits of 90-95 kDa. In humans, it isexpressed at high levels on vascular endothelial cells and onsyncytiotrophoblast of term placenta. During human heart development, itis expressed at high levels on endocardial cushion tissue mesenchymeduring heart septation and valve formation; subsequently expressiondrops as the valves mature. It also is expressed by a population ofpre-erythroblasts, leukemic cells of lymphoid and myeloid lineages, andbone marrow stromal fibroblasts. Endoglin is an accessory protein ofmultiple kinase receptor complexes of the TGF-β superfamily. The TGF-β1superfamily of structurally related peptides includes the TGF-βisoforms, β1, β2, β3, and β5, the activins and the bone morphogeneticproteins (BMPs). TGF-β-like factors are a multifunctional set ofconserved growth and differentiation factors that control biologicalprocesses such as embryogenesis, organogenesis, morphogenesis of tissueslike bone and cartilage, vasculogenesis, wound repair and angiogenesis,hematopoiesis, and immune regulation. Signaling by ligands of the TGF-βsuperfamily is mediated by a high affinity, ligand-induced, heteromericcomplex consisting of related Ser/Thr kinase receptors divided into twosubfamilies, type I and type II. The type II receptortransphosphorylates and activates the type I receptor in a Gly/Ser-richregion. The type I receptor in turn phosphorylates and transducessignals to a novel family of recently identified downstream targets,termed Smads. Endoglin binds transforming growth factor (TGF) TGF-β1 and-β3 by associating with the TGF-β type II receptor, interacts withactivin-A, interacts with bone morphogenic protein (BMP)-7 via activintype II receptors, ActRII and ActRIIB, and binds BMP-2 by interactingwith the ligand binding type I receptors ALK3 and ALK6.

CD166 antigen (ALCAM), a 556 amino acid glycoprotein belonging to theimmunoglobulin gene superfamily, is encoded by the activatedleukocyte-cell adhesion molecule (ALCAM) gene in humans. It contains asecretory signal sequence, an extracellular domain which contains 3Ig-like C2-type domains, 2 Ig-like V-type domains and 9 potentialN-linked glycosylation sites, a hydrophobic transmembrane spanningdomain and a 32 amino acid cytoplasmic domain with no known motifs. TheN-terminal Ig domain is the binding site for both homophilic andCD166-CD6 interactions. CD166 is anchored to the actin cytoskeleton viathe cytoplasmic domain but the receptors involved in this interactionare unknown. The soluble CD166 is produced by proteolytic cleavage ofextracellular domains or by alternative splicing. It is expressed onmesenchymal stem cells and progenitor cells and on cortical thymicepithelial cells and medullary thymic epithelial cells, neurons,activated T cells, B cells, monocytes, fibroblasts, endothelium,epithelium, primitive subsets of hematopoietic cells includingpluripotent stem cells, blastocysts and endometrium.

The term “CXCR-4” as used herein refers to a G-protein-linked chemokinereceptor. Stromal-derived factor-1 (SDF-1), an alpha-chemokine thatbinds to G-protein-coupled CXCR4, plays an important role in theregulation of stem/progenitor cell trafficking.

The term “cell” is used herein to refer to the structural and functionalunit of living organisms and is the smallest unit of an organismclassified as living.

The term “chemokine” as used herein refers to a class of chemotacticcytokines that signal leukocytes to move in a specific direction.

The terms “chemotaxis” or “chemotactic” refer to the directed motion ofa motile cell or part along a chemical concentration gradient towardsenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

The term “chondrocytes” as used herein refers to cells found incartilage that produce and maintain the cartilaginous matrix for, forexample, joints, ear canals, trachea, epiglottis, larynx, the discsbetween vertebrae and the ends of ribs. From least to terminallydifferentiated, the chondrocytic lineage is (i) Colony-formingunit-fibroblast (CFU-F); (ii) mesenchymal stem cell/marrow stromal cell(MSC); (iii) chondrocyte.

The term “chondrogenesis” as used herein refers to the formation of newcartilage from cartilage forming or chondrocompetent cells.

The term “chondrogenic” as used herein refers to a potential ofundifferentiated precursor cells to differentiate into cartilage formingor chondrocompetent cells cells.

The term “compatible” as used herein means that the components of acomposition are capable of being combined with each other in a mannersuch that there is no interaction that would substantially reduce theefficacy of the composition under ordinary use conditions.

The term “component” as used herein refers to a constituent part,element or ingredient.

The term “condition”, as used herein, refers to a variety of healthstates and is meant to include disorders or diseases caused by anyunderlying mechanism or disorder, injury, and the promotion of healthytissues and organs.

The term “contact” and its various grammatical forms as used hereinrefers to a state or condition of touching or of immediate or localproximity. Contacting a composition to a target destination may occur byany means of administration known to the skilled artisan.

The term “cut section thickness” as used herein refers to thickness of asection as measured directly from the sectioning device (cryostat,microtome, etc.) prior to histological processing, which may causeshrinkage in the z-axis. Also known as the block advance of themicrotome.

The term “cytokine” as used herein refers to small soluble proteinsubstances secreted by cells which have a variety of effects on othercells. Cytokines mediate many important physiological functionsincluding growth, development, wound healing, and the immune response.They act by binding to their cell-specific receptors located in the cellmembrane, which allows a distinct signal transduction cascade to startin the cell, which eventually will lead to biochemical and phenotypicchanges in target cells. Generally, cytokines act locally. They includetype I cytokines, which encompass many of the interleukins, as well asseveral hematopoietic growth factors; type II cytokines, including theinterferons and interleukin-10; tumor necrosis factor (“TNF”)-relatedmolecules, including TNFα and lymphotoxin; immunoglobulin super-familymembers, including interleukin 1 (“IL-1”); and the chemokines, a familyof molecules that play a critical role in a wide variety of immune andinflammatory functions. The same cytokine can have different effects ona cell depending on the state of the cell. Cytokines often regulate theexpression of, and trigger cascades of other cytokines Nonlimitingexamples of cytokines include e.g., IL-1.alpha., IL-.beta., IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12/IL-23 P40, IL13,IL-17, IL-18, TGF-beta., IFN-gamma., GM-CSF, Gro.alpha., MCP-1 andTNF-alpha.

The term “cytometry” as used herein refers to a process in whichphysical and/or chemical characteristics of single cells, or byextension, of other biological or nonbiological particles in roughly thesame size or stage, are measured. In flow cytometry, the measurementsare made as the cells or particles pass through the measuring apparatus(a flow cytometer) in a fluid stream. A cell sorter, or flow sorter, isa flow cytometer that uses electrical and/or mechanical means to divertand collect cells (or other small particles) with measuredcharacteristics that fall within a user-selected range of values.

“Demineralized bone matrix” (DBM) refers to a bone-derived material thathas osteoconductive and osteoinductive activity. DBM may be prepared byacid extraction of allograft bone, resulting in loss of most of themineralized component but retention of collagen and noncollagenousproteins, including growth factors. Methods for preparing demineralizedbone matrix from bone are known in the art, as disclosed, for example,in U.S. Pat. Nos. 5,073,373; 5,484,601; and 5,284,655, which areincorporated herein by reference. DBM may be prepared from autologousbone, allogeneic (or “allograft”) bone, or xenogeneic bone. DBM may beprepared from cancellous bone, cortical bone, or combinations ofcancellous and cortical bone. For the purpose of the present disclosure,demineralized bone includes bone matrix having a residual mineralcontent of 5% or less (w/w), 2% or less (w/w), 1% or less (w/w), 0.5% orless (w/w), or consisting essentially of collagen, non-collagen proteinssuch as growth factors, and other nonmineral substances found in theoriginal bone, although not necessarily in their original quantities.The term “demineralized cortical bone” (DCB) as used herein refers to ademineralized allograft cortical bone

The term “derivative” as used herein means a compound that may beproduced from another compound of similar structure in one or moresteps. A “derivative” or “derivatives” of a peptide or a compoundretains at least a degree of the desired function of the peptide orcompound. Accordingly, an alternate term for “derivative” may be“functional derivative.” Derivatives can include chemical modificationsof the peptide, such as akylation, acylation, carbamylation, iodinationor any modification that derivatizes the peptide. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formal groups. Free carboxyl groups can bederivatized to form salts, esters, amides, or hydrazides. Free hydroxylgroups can be derivatized to form O-acyl or O-alkyl derivatives. Theimidazole nitrogen of histidine can be derivatized to formN-im-benzylhistidine. Also included as derivatives or analogues arethose peptides that contain one or more naturally occurring amino acidderivative of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamiate, and can include amino acids that are notlinked by peptide bonds. Such peptide derivatives can be incorporatedduring synthesis of a peptide, or a peptide can be modified bywell-known chemical modification methods (see, e.g., Glazer et al.,Chemical Modification of Proteins, Selected Methods and AnalyticalProcedures, Elsevier Biomedical Press, New York (1975)).

The term “detectable marker” encompasses both selectable markers andassay markers. The term “selectable markers” refers to a variety of geneproducts to which cells transformed with an expression construct can beselected or screened, including drug-resistance markers, antigenicmarkers useful in fluorescence-activated cell sorting, adherence markerssuch as receptors for adherence ligands allowing selective adherence,and the like.

The term “detectable response” refers to any signal or response that maybe detected in an assay, which may be performed with or without adetection reagent. Detectable responses include, but are not limited to,radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared,visible) emission, absorption, polarization, fluorescence,phosphorescence, transmission, reflection or resonance transfer.Detectable responses also include chromatographic mobility, turbidity,electrophoretic mobility, mass spectrum, ultraviolet spectrum, infraredspectrum, nuclear magnetic resonance spectrum and x-ray diffraction.Alternatively, a detectable response may be the result of an assay tomeasure one or more properties of a biologic material, such as meltingpoint, density, conductivity, surface acoustic waves, catalytic activityor elemental composition. A “detection reagent” is any molecule thatgenerates a detectable response indicative of the presence or absence ofa substance of interest. Detection reagents include any of a variety ofmolecules, such as antibodies, nucleic acid sequences and enzymes. Tofacilitate detection, a detection reagent may comprise a marker.

The term “differential label” as used herein generally refers to astain, dye, marker, or antibody used to characterize or contraststructures, components or proteins of a single cell or organism.

The term “differentiation” as used herein refers to the process ofdevelopment with an increase in the level of organization or complexityof a cell or tissue, accompanied with a more specialized function.

The terms “disease” or “disorder” as used herein refer to an impairmentof health or a condition of abnormal functioning.

The term “dye” (also referred to as “fluorochrome” or “fluorophore”) asused herein refers to a component of a molecule which causes themolecule to be fluorescent. The component is a functional group in themolecule that absorbs energy of a specific wavelength and re-emitsenergy at a different (but equally specific) wavelength. The amount andwavelength of the emitted energy depend on both the dye and the chemicalenvironment of the dye. Many dyes are known, including, but not limitedto, FITC, R-phycoerythrin (PE), PE-Texas Red Tandem, PE-Cy5 Tandem,propidium iodem, EGFP, EYGP, ECF, DsRed, allophycocyanin (APC), PerCp,SYTOX Green, courmarin, Alexa Fluors (350, 430, 488, 532, 546, 555, 568,594, 633, 647, 660, 680, 700, 750), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7,Hoechst 33342, DAPI, Hoechst 33258, SYTOX Blue, chromomycin A3,mithramycin, YOYO-1, SYTOX Orange, ethidium bromide, 7-AAD, acridineorange, TOTO-1, TO-PRO-1, thiazole orange, TOTO-3, TO-PRO-3, thiazoleorange, propidium iodide (PI), LDS 751, Indo-1, Fluo-3, DCFH, DHR,SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP, GFP, AmCyanl, Y77W, S65A, S65C,S65L, S65T, ZsGreen1, ZsYellow1, DsRed2, DsRed monomer, AsRed2, mRFP1,HcRed1, monochlorobimane, calcein, the DyLight Fluors, cyanine,hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, LuciferYellow, NBD, PE-Cy5 conjugates, PE-Cy7 conjugates, APC-Cy7 conjugates,Red 613, fluorescein, FluorX, BODIDY-FL, TRITC, X-rhodamine, LissamineRhodamine B, Texas Red, TruRed, and derivatives thereof.

The term “nonexpanded” as used herein refers to a cell population thathas not been grown in culture (in vitro) to increase the number of cellsin the cell population.

The term “endogenous” as used herein refers to that which is naturallyoccurring, incorporated within, housed within, adherent to, attached toor resident in.

The term “extracellular matrix” as used herein refers to a scaffold in acell's external environment with which the cell interacts via specificcell surface receptors. The extracellular matrix serves many functions,including, but not limited to, providing support and anchorage forcells, segregating one tissue from another tissue, and regulatingintracellular communication. The extracellular matrix is composed of aninterlocking mesh of fibrous proteins and glycosaminoglycans (GAGs).Examples of fibrous proteins found in the extracellular matrix includecollagen, elastin, fibronectin, and laminin. Examples of GAGs found inthe extracellular matrix include proteoglycans (e.g., heparin sulfate),chondroitin sulfate, keratin sulfate, and non-proteoglycanpolysaccharide (e.g., hyaluronic acid). The term “proteoglycan” refersto a group of glycoproteins that contain a core protein to which isattached one or more glycosaminoglycans.

The term “factors” as used herein refers to nonliving components thathave a chemical or physical effect. For example, a “paracrine factor” isa diffusible signaling molecule that is secreted from one cell type thatacts on another cell type in a tissue. A “transcription factor” is aprotein that binds to specific DNA sequences and thereby controls thetransfer of genetic information from DNA to mRNA.

The term “fluorescence” as used herein refers to the result of athree-state process that occurs in certain molecules, generally referredto as “fluorophores” or “fluorescent dyes,” when a molecule ornanostructure relaxes to its ground state after being electricallyexcited. Stage 1 involves the excitation of a fluorophore through theabsorption of light energy; Stage 2 involves a transient excitedlifetime with some loss of energy; and Stage 3 involves the return ofthe fluorophore to its ground state accompanied by the emission oflight.

The term “fluorescent-activated cell sorting” (also referred to as“FACS”) as used herein refers to a method for sorting a heterogeneousmixture of biological cells into one or more containers, one cell at atime, based upon the specific light scattering and fluorescentcharacteristics of each cell.

The term “fossa” as used herein means a small cavity or depression, asin a bone.

The term “fragment” as used herein refers to a small part, which may be,without exclusion, a particle, chip, or fiber, derived from, cut off, orbroken from a larger unit which retains the desired biological activityof the larger unit.

The term “functional equivalent” or “functionally equivalent” are usedinterchangeably herein to refer to substances, molecules,polynucleotides, proteins, peptides, or polypeptides having similar oridentical effects or use.

The term “graft” as used herein refers to a tissue or organ transplantedfrom a donor to a recipient. It includes, but is not limited to, a selftissue transferred from one body site to another in the same individual(“autologous graft”), a tissue transferred between genetically identicalindividuals or sufficiently immunologically compatible to allow tissuetransplant (“syngeneic graft”), a tissue transferred between geneticallydifferent members of the same species (“allogeneic graft” or“allograft”), and a tissue transferred between different species(“xenograft”).

The term “growth” as used herein refers to a process of becoming larger,longer or more numerous, or an increase in size, number, or volume.

The term “growth conduction” as used herein refers to a process by whicha tissue is directed to regenerate or grow so as to conform to amaterial's surface. A growth-conductive surface is one that permitstissue growth on its surface or down into pores, channels or pipes.Growth-conductive material facilitates the spontaneous formation of atissue by furnishing a microenvironment that supports deposition oradhesion of tissuegenic cells and optionally, vascularization. Examplesof growth-conductive materials, include, but are not limited to,processed human bone (e.g., allograft bone, which may be anosteoconductive material), purified collagen, calcium phosphateceramics, synthetic polymers, tissue-derived matrices, BMP-2 and 4,VEGF, bFGF, TGF-β, and PDGF.

The term “growth-conductive matrix” as used herein refers to a matrixthat may be inert in and of itself but which supports three-dimensionaltissue formation. For example, allograft bone tissue may be anosteoconductive matrix.

The term “growth factor” as used herein refers to extracellularpolypeptide molecules that bind to a cell-surface receptor triggering anintracellular signaling pathway, leading to proliferation,differentiation, or other cellular response. Growth factors include, butare not limited to, cytokines and hormones.

The term “growth induction” as used herein refers to a process by whichprimitive, undifferentiated and tissuegenic cells are stimulated todevelop into an ensemble of cells, not necessarily identical, thattogether carry out a specific function. This ensemble of cells is termeda tissue.

The term “growth-inductive matrix” as used herein refers to a matrixcontaining a substance or substances capable of recruiting orstimulating local tissuegenic cells so that the cells are induced(meaning to cause, bring about, bring about, or trigger) todifferentiate and/or produce a tissue.

The terms “growth-inductive components” or “growth-inductive factors” or“tissuegenic factors” are used interchangeably to refer to the plethoraof mediators associated with tissue development and repair.

For example, Table 11 lists exemplary growth-inductive factors secretedby adipose tissue classified according to metabolic, immunological orother function. (Halberg et. al., 2008, Endocrinol. Metab. Clin. NorthAm., 37(3): 753-767). The subcutaneous adipose secretome includesadiponectin, leptin, IL-6, IL-7, IL-8, MCP-1, GRO, angiogenin, HGF,VEGF, TIMP-1, TIMP-2, etc. (Klimkakova et. al., 2007, Biochem. Biophys.Res. Commun., 358: 897-902).

TABLE 11 Secreted Soluble non-ECM Factors of Adipose Secretome MetabolicFactors Immunological Factors Other Factors Adipsin Alpha 1 acidglycoprotein Angiogenin Adiponectin Colony stimulating factor-1Angiopoietin 1 Apelin Complement component Angiopoietin 2 inhibitor C1ApoE Complement C1 Angiotensinogen Cortisol Complement C2 CalcitoninInsulin-like growth factor 1 Complement C3 Chemerin (IGF-1) Insulin-likegrowth factor Complement C4 Cyclophilin A (IGF) Binding protein 7 (Bp 7)Complement C7 Extracellular SOD Lipoprotein lipase Complement factor BGalectin 1 Leptin Complement factor C Growth related oncogene (GRO)Fasting induced adipose Complement factor D Fibroblast growth factorfactor (FGF) Plasminogen activated C reactive protein Hepatic growthfactor (GF) inhibitor −1 Resistin Haptoglobin Mineralcorticoid releasingfactor (MRF) Retinol binding protein 4 Interleukin 1 beta (IL-1β)Monocyte chemoattractant protein 1 (MCP-1) Vaspin Interleukin 4 (IL-4)Nerve growth factor (NGF) Vistafin Interleukin 6 (IL-6) Pigmentepithelium derived factor (PEDF) Interleukin 7 (IL-7) Prostaglandin E2Interleukin 8 (IL-8 Prostaglandin I2 Interleukin 10 (IL-10)Prostaglandin 2alpha Interleukin 12 (IL-12) Serum transferringInterleukin 18 (IL-18) Stromal derived factor 1 Lipocalin 24p3 TGF betaMacrophage migration TIMP-1 inhibitory factor 1 Serum amyloid A3 (SAA3)TIMP-2 Tumor necrosis factor Tissue factor alpha (TNF-α) Vascularendothelial growth factor (VEGF)

The term “hematopoietic stem cell” refers to a cell isolated from theblood or from the bone marrow that can renew itself, differentiate to avariety of specialized cells, mobilize out of the bone marrow into thecirculating blood, and undergo programmed cell death (apoptosis). Insome embodiments of the described invention, hematopoietic stem cellsderived from human subjects express at least one type of cell surfacemarker, including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59,Sca-1, Thy-1, and/or CXCR-4, or a combination thereof.

“HLA-DR” refers to a human class II histocompatibility antigen presenton several cell types, including antigen-presenting cells, B cells,monocytes, macrophages, and activated T cells.

The term “interleukin” as used herein refers to a cytokine secreted bywhite blood cells as a means of communication with other white bloodcells.

The term “implant” refers to any device or material inserted or placed,permanently or temporarily, into or onto a subject as well as those usedfor the administration or delivery of a therapeutic agent(s) orsubstance.

The term “improve” (or improving) as used herein refers to bring into amore desirable or excellent condition.

The terms “in the body”, “void volume”, “resection pocket”,“excavation”, “injection site”, “deposition site” or “implant site” asused herein are meant to include all tissues of the body without limit,and may refer to spaces formed therein from injections, surgicalincisions, tumor or tissue removal, tissue injuries, abscess formation,or any other similar cavity, space, or pocket formed thus by action ofclinical assessment, treatment or physiologic response to disease orpathology as non-limiting examples thereof.

The term “indicator” as used herein refers to any substance, number orratio derived from a series of observed facts that may reveal relativechanges as a function of time; or a signal, sign, mark, note or symptomthat is visible or evidence of the existence or presence thereof.

The term “inflammation” as used herein refers to the physiologic processby which vascularized tissues respond to injury. See, e.g., FUNDAMENTALIMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers,Philadelphia (1999) at 1051-1053, incorporated herein by reference.During the inflammatory process, cells involved in detoxification andrepair are mobilized to the compromised site by inflammatory mediators.Inflammation is often characterized by a strong infiltration ofleukocytes at the site of inflammation, particularly neutrophils(polymorphonuclear cells). These cells promote tissue damage byreleasing toxic substances at the vascular wall or in uninjured tissue.Traditionally, inflammation has been divided into acute and chronicresponses.

The term “acute inflammation” as used herein refers to the rapid,short-lived (minutes to days), relatively uniform response to acuteinjury characterized by accumulations of fluid, plasma proteins, andneutrophilic leukocytes. Examples of injurious agents that cause acuteinflammation include, but are not limited to, pathogens (e.g., bacteria,viruses, parasites), foreign bodies from exogenous (e.g. asbestos) orendogenous (e.g., urate crystals, immune complexes), sources, andphysical (e.g., burns) or chemical (e.g., caustics) agents.

The term “chronic inflammation” as used herein refers to inflammationthat is of longer duration and which has a vague and indefinitetermination. Chronic inflammation takes over when acute inflammationpersists, either through incomplete clearance of the initialinflammatory agent or as a result of multiple acute events occurring inthe same location. Chronic inflammation, which includes the influx oflymphocytes and macrophages and fibroblast growth, may result in tissuescarring at sites of prolonged or repeated inflammatory activity.

The term “injury,” as used herein, refers to damage or harm to astructure or function of the body caused by an outside agent or force,which may be physical or chemical.

The term “isolate” and its various grammatical forms as used hereinrefers to placing, setting apart, or obtaining a protein, molecule,substance, nucleic acid, peptide, cell or particle, in a formessentially free from contaminants or other materials with which it iscommonly associated, separate from its natural environment.

The term “labeling” as used herein refers to a process of distinguishinga compound, structure, protein, peptide, antibody, cell or cellcomponent by introducing a traceable constituent. Common traceableconstituents include, but are not limited to, a fluorescent antibody, afluorophore, a dye or a fluorescent dye, a stain or a fluorescent stain,a marker, a fluorescent marker, a chemical stain, a differential stain,a differential label, and a radioisotope.

The term “labile” as used herein refers to subject to increaseddegradation.

The terms “marker” or “cell surface marker” are used interchangeablyherein to refer to an antigenic determinant or epitope found on thesurface of a specific type of cell. Cell surface markers can facilitatethe characterization of a cell type, its identification, and eventuallyits isolation. Cell sorting techniques are based on cellular biomarkerswhere a cell surface marker(s) may be used for either positive selectionor negative selection, i.e., for inclusion or exclusion, from a cellpopulation.

The term “matrix” refers to a surrounding substance within whichsomething is contained or embedded.

The term “mechanical agitation” as used herein refers to a processwhereby tissue is physically shaken or churned via mechanical means.Such mechanical means include, but are not limited to, a mixer or othermechanical device.

The term “mesenchymal stem cells (MSCs)” as used herein refers tonon-blood adult stem cells found in a variety of tissues. They arecharacterized by their spindle-shape morphologically; by the expressionof specific markers on their cell surface; and by their ability underappropriate conditions, to differentiates along a minimum of threelineages (osteogenic, chondrogenic and adipogenic). When referring tobone or cartilage, MSCs commonly are known as osteochondrogenic,osteogenic, or chondrogenic, since a single MSC has shown the ability todifferentiate into chondrocytes or osteoblasts, depending on the medium.

MSCs secrete many biologically important molecules, includinginterleukins 6, 7, 8, 11, 12, 14, and 15, M-CSF, Flt-3 ligand, SCF, LIF,bFGF, VEGF, P1GF and MCP1 (Majumdar, et al., J. Cell Physiol. 176: 57-66(1998), Kinnaird et al, Circulation 109: 1543-49 (2004)). In 2004, itwas reported that no single marker that definitively identifies MSCs invivo had yet been identified, due to the lack of consensus from diversedocumentations of the MSC phenotype. Baksh, et al., J. Cell. Mol. Med.8(3): 301-16, 305 (2004). There is general agreement that MSCs lacktypical hematopoietic antigens, namely CD14, CD34, and CD45. (Id.;citing Pittenger, M. F. et al., Science 284: 143-47 (1999)).

The term “mill,” and its various grammatical forms, as used hereinrefers to grind, to cut, to shred, to chip, or to pulverize a substance.

The term “mounted section thickness” as used herein, refers to thethickness of tissue sections after histological processing.

The term “multipotent” as used herein refers to a cell capable of givingrise to a limited number of cell types of a particular cell line.

The term “myogenic” refers to a potential of undifferentiated precursorcells to differentiate into a muscle forming or myocompetent cells.

The term “Optical Disector” refers to a stereological probe for countingor selecting objects in a tissue section. This is an extension to thebasic Disector method, which is applied to a thick section using aseries, or stack, of Disectors. Rather than using pairs of physicalsections (the basic Disector method), optical sectioning is used bycreating focal planes with a thin depth-of-field through the section.The Optical Disector begins with a lookup section at the top of theoptical disector and ends with a reference section at the bottom of theoptical disector. The focal plane is the current reference section. Thelookup section is immediately above the focal plane. A particle in focusat the top of the optical disector is therefore seen in the lookupsection and not counted. A particle in focus at the bottom of theoptical disector is in the reference section and therefore not in thelookup section, is counted. Counting frame rules are applied when theparticle first comes into focus.

The term “osteoblasts” as used herein refers to cells that arise whenosteoprogenitor cells or mesenchymal cells, which are located near allbony surfaces and within the bone marrow, differentiate under theinfluence of growth factors. Osteoblasts, which are responsible for bonematrix synthesis, secrete a collagen rich ground substance essential forlater mineralization of hydroxyapatite and other crystals. The collagenstrands to form osteoids (spiral fibers of bone matrix). Osteoblastscause calcium salts and phosphorus to precipitate from the blood, whichbond with the newly formed osteoid to mineralize the bone tissue. Onceosteoblasts become trapped in the matrix they secrete, they becomeosteocytes. From least to terminally differentiated, the osteocytelineage is (i) Colony-forming unit-fibroblast (CFU-F); (ii) mesenchymalstem cell/marrow stromal cell (MSC); (iii) osteoblast; and (iv)osteocyte.

The term “osteocalcin” as used herein refers to a protein constituent ofbone; circulating levels are used as a marker of increased boneturnover.

The term “osteoclast” as used herein refers to large multinucleate cellsassociated with areas of bone resorption (breakdown).

The term “osteoconduction” as used herein refers to a process by whichbone is directed so as to conform to a material's surface. Anosteoconductive surface is one that permits bone growth on its surfaceor down into pores, channels or pipes. Osteoconductive materialfacilitates the spontaneous formation of bone by furnishing amicroenvironment that supports the ingrowth of blood vessels,perivascular tissue and osteoprogenitor cells into the site where it isdeposited. Examples of osteoconductive materials, include, but notlimited to, processed human bone (allograft bone), purified collagen,calcium phosphate ceramics, synthetic polymers, BMP-2 and 4, VEGF, bFGF,TGF-β, and PDGF.

The term “osteoconductive matrix” as used herein refers to a matrix thatis inert in and of itself but on which cells can climb and grow bone.

The term “osteogenic” refers to a potential of undifferentiatedprecursor cells to differentiate into bone forming or osteocompetentcells.

The term “osteogenesis” as used herein refers to the development orformation of new bone by bone forming or osteocompetent cells.

The term “osteoinduction” as used herein refers to a process by whichprimitive, undifferentiated and pluripotent cells are stimulated todevelop into a bone forming cell lineage thereby inducing osteogenesis.For example, the majority of bone healing in a fracture is dependent onosteoinduction. Osteoinductive materials can be generated by combining aporous scaffold with osteogenic cells and/or osteoinductive components,including, but not limited to, growth factors such as BMP-2 and 4, VEGF,bFGF, TGF-β, and PDGF.

The term “osteoinductive matrix” as used herein refers to a matrixcontaining a substance or substances that recruit local cells to induce(meaning to cause, bring about, bring about, or trigger) local cells toproduce bone.

The terms “osteoinductive components” or “osteogenic factors” are usedinterchangeably to refer to the plethora of mediators associated withbone development and repair, including, but not limited to, bonemorphogenic proteins (BMPs), vascular endothelial growth factor (VEGF),basic fibroblast growth factor (bFGF), transforming growth factor beta(TGFβ), and platelet-derived growth factor (PDGF).

The term “osteointegration” refers to an anchorage mechanism wherebynonvital components can be incorporated reliably into living bone andthat persist under all normal conditions of loading.

The term “particle” as used herein refers to a chip, fragment, slice,fiber or other small constituent of a larger body (e.g., picoparticles,nanoparticles, microparticles, milliparticle, centiparticle,deciparticle; fractions thereof, or, in some instances, a larger segmentor piece).

The term “piece” as used herein refers to a particle, section, strip,chip, fragment, slice, fiber or other part, derived from, cut off, orbroken from a larger unit.

The term “peptide” is used herein to refer to two or more amino acidsjoined by a peptide bond.

The term “periosteum” as used herein refers to the normal investment ofbone, consisting of a dense, fibrous outer layer, to which musclesattach, and a more delicate, inner layer capable of forming bone.

The term “Platelet Derived Growth Factor” (PDGF) as used herein refersto a major mitogen for connective tissue cells and certain other celltypes. It is a dimeric molecule consisting of disulfide-bonded,structurally similar A and B-polypeptide chains, which combine to homo-and hetero-dimers. The PDGF isoforms exert their cellular effects bybinding to and activating two structurally related protein tyrosinekinase receptors, the α-receptor and the β-receptor. Activation of PDGFreceptors leads to stimulation of cell growth, but also to changes incell shape and motility; PDGF induces reorganization of the actinfilament system and stimulates chemotaxis, i.e., a directed cellmovement toward a gradient of PDGF. In vivo, PDGF plays a role inembryonic development and during wound healing.

The term “pluripotent” as used herein refers to the ability to developinto multiple cells types, including all three embryonic lineages,forming the body organs, nervous system, skin, muscle and skeleton.

The term “progenitor cell” as used herein refers to an early descendantof a stem cell that can only differentiate, but can no longer renewitself. Progenitor cells mature into precursor cells that mature intomature phenotypes. Hematopoietic progenitor cells are referred to ascolony-forming units (CFU) or colony-forming cells (CFC). The specificlineage of a progenitor cell is indicated by a suffix, such as, but notlimited to, CFU-E (erythrocytic), CFU-F (fibroblastic), CFU-GM(granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoieticprogenitor). Osteoclasts arise from hematopoietic cells of themonocyte/neutrophil lineage (CFU-GM). Osteoprogenitor cells arise frommesenchymal stem cells and are committed to an osteocyte lineage.

The term “propagate” as used herein refers to reproduce, multiply, or toincrease in number, amount or extent by any process.

The term “purification” as used herein refers to the process ofisolating or freeing from foreign, extraneous, or objectionableelements.

The term “random” as used herein refers to unpredictable. There is someelement of chance. This is the opposite of deterministic, in which thenext number or event is knowable.

The term “reduced” or “to reduce” as used herein refers to adiminishing, a decrease in, an attenuation or abatement of the degree,intensity, extent, size, amount, density or number of.

The term “regeneration” or “regenerate” as used herein refers to aprocess of recreation, reconstitution, renewal, revival, restoration,differentiation and growth to form a tissue with characteristics thatconform with a natural counterpart of the tissue.

The term “relative” as used herein refers to something having, orstanding in, some significant association to something else. The term“relative frequency” as used herein refer to the rate of occurrence ofsomething having or standing in some significant association to the rateof occurrence of something else. For example, two cell types, X cellsand Y cells occupy a given location. There are 5× cells and 5 Y cells inthat location. The relative frequency of cell type X is 5/10; therelative frequency of cell type Y is 5/10 in that location. Followingprocessing, there are 5× cells, but only 1 Y cell in that location. Therelative frequency of cell type X following processing is 5/6, and therelative frequency of cell type Y following processing is 1/6 in thatlocation.

The term “repair” as used herein as a noun refers to any correction,reinforcement, reconditioning, remedy, making up for, making sound,renewal, mending, patching, or the like that restores function. Whenused as a verb, it means to correct, to reinforce, to recondition, toremedy, to make up for, to make sound, to renew, to mend, to patch or tootherwise restore function. In some embodiments “repair” includes fullrepair and partial repair.

The term “resident,” and its various grammatical forms, as used hereinrefers to being present habitually, existing in or intrinsic to orincorporated therein.

The term “rinse,” and its various grammatical forms, as used hereinrefers to wash, to douse with a liquid or liquids or to flow a liquid orliquids over the material being rinsed.

The term “scaffold” as used herein refers to a structure capable ofsupporting a three-dimensional tissue formation. A three-dimensionalscaffold is believed to be critical to replicate the in vivo milieu andto allow the cells to influence their own microenvironment. Scaffoldsmay serve to promote cell attachment and migration, to deliver andretain cells and biochemical factors, to enable diffusion of vital cellnutrients and expressed products, and to exert certain mechanical andbiological influences to modify the behavior of the cell phase. Ascaffold utilized for tissue reconstruction has several requisites. Sucha scaffold should have a high porosity and an adequate pore size tofacilitate cell seeding and diffusion of both cells and nutrientsthroughout the whole structure. Biodegradability of the scaffold is alsoan essential requisite. The scaffold should be absorbed by thesurrounding tissues without the necessity of a surgical removal, suchthat the rate at which degradation occurs coincides as closely aspossible with the rate of tissue formation. As cells are fabricatingtheir own natural matrix structure around themselves, the scaffoldprovides structural integrity within the body and eventually degradesleaving the neotissue (newly formed tissue) to assume the mechanicalload.

The term “section” when used in the context of stereology refers to acut through material that has effectively zero thickness compared to thesize of the particles being studied. Biologists refer to sections asthick slices through tissue. The actual thickness of sections can leadsto the Holmes effect.

The term “side-effect” as used herein refers to a result of a therapy inaddition to, or in extension of, the desired therapeutic effect.

The term “similar” is used interchangeably with the terms analogous,comparable, or resembling, meaning having traits or characteristics incommon.

A “solution” generally is considered as a homogeneous mixture of two ormore substances. It is frequently, though not necessarily, a liquid. Ina solution, the molecules of the solute (or dissolved substance) areuniformly distributed among those of the solvent. The term “solvent” asused herein refers to a substance capable of dissolving anothersubstance (termed a “solute”) to form a uniformly dispersed mixture(solution).

The term “stain” as used herein refers to a composition of a dye(s) orpigment(s) used to make a structure, a material, a cell, a cellcomponent, a membrane, a granule, a nucleus, a cell surface receptor, apeptide, a microorganism, a nucleic acid, a protein or a tissuedifferentiable.

The term “Sca-1” or “stem cell antigen-1” refers to a surface proteincomponent in a signaling pathway that affects the self-renewal abilityof mesenchymal stem cells.

The term “stem cells” refers to undifferentiated cells having highproliferative potential with the ability to self-renew (make more stemcells by cell division) that can generate daughter cells that canundergo terminal differentiation into more than one distinct cellphenotype.

The term “stereology” as used herein refers to a method of quantifying2D and 3D structures using estimation methods.

The term “stimulate” as used herein refers to activate, provoke, orspur. The term “stimulating agent” as used herein refers to a substancethat exerts some force or effect.

The phrase “subject in need thereof” as used herein refers to a patientthat (i) will be administered at least one allograft, (ii) is receivingat least one allograft; or (iii) has received at least one allograft,unless the context and usage of the phrase indicates otherwise.

The term “substantially similar” as used herein means that a firstvalue, aspect, trait, feature, number, or amount is of at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofa second value, aspect, trait, feature, number, or amount.

The term “surfactant”, as used herein, refers to a surface-active agentthat acts to reduce surface tension, which is the elastic like forceexisting in the surface of a body, e.g., a liquid, at an interfacebetween two liquids, or that between a liquid and a solid, tending tominimize the area of the surface, caused by asymmetries in theintermolecular forces between surface molecules. Surfactants usually areorganic compounds that contain both hydrophobic groups and hydrophilicgroups, i.e., are amphiphilic. Surfactants can be anionic, cationic,nonionic, and zwitterionic. Exemplary surfactants include, but are notlimited to, Triton®, Tween® 80, egg lecithin, vitamin E-t d-α-tocopherylpolyethylene glycol 1000 succinate (TPGS). Exemplary surfactantssuitable for use in this invention are described in, for example,Becher, Emulsions Theory and Practice; Robert E. Krieger Publishing,Malabar, Fla. (1965), which is incorporated herein by reference.

The term “symptom” as used herein refers to a sign or an indication ofdisorder or disease, especially when experienced by an individual as achange from normal function, sensation, or appearance.

The term “therapeutic effect” as used herein refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect may include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect also may include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.

The term “tissuegenic” as used herein refers to a potential of anundifferentiated precursor cell to differentiate into a mature cell typeand to regenerate a tissue. Exemplary tissuegenic cells include but arenot limited to a stem cell, a progenitor cell or a combination thereof.The term “osteogenic” refers more specifically to cell differentiationand tissue regeneration with regard to bone.

The term “transforming growth factor beta (TGFβ) signaling pathway” isused herein to refer to the signaling pathway is involved in manycellular processes in both the adult organism and the developing embryoincluding cell growth, cell differentiation, apoptosis, cellularhomeostasis and other cellular functions. TGFβ superfamily ligands bindto a type II receptor, which recruits and phosphorylates a type Ireceptor. The type I receptor then phosphorylates receptor-regulatedSMADs (R-SMADs) which can now bind the coSMAD SMAD4. R-SMAD/coSMADcomplexes accumulate in the nucleus where they act as transcriptionfactors and participate in the regulation of target gene expression.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. Treating further refers toaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting development of symptoms characteristic of thedisorder(s) being treated; (c) limiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting recurrence of symptoms in patients that were previouslyasymptomatic for the disorder(s).

The term “vascularization” as used herein refers to a process ofingrowth of blood vessels and perivascular tissue within agrowth-conductive matrix to support the deposition and adhesion oftissuegenic cells to effect tissue regeneration.

The terms “VEGF”, “VEGF-1” or “vascular endothelial growth factor-1” areused interchangeably herein to refer to a cytokine that mediatesnumerous functions of endothelial cells including proliferation,migration, invasion, survival, and permeability. The term “VEGF-2”refers to a regulator for growth of vascular endothelial and smoothmuscle cells. VEGF-2 stimulates the growth of human vascular endothelialcells but inhibits growth of human aortic smooth muscle cells induced byplatelet-derived growth factor.

The term “viable” as used herein refers to having the ability to grow,expand, or develop; capable of living.

The term “xenogeneic” as used herein refers to cells or tissues derivedfrom individuals of different species, including, but not limited to,porcine, bovine, caprine, equine, canine, lapine, feline, and/ornon-human mammals, such as, but not limited to, whale, and porpoise.

1. Implant

According to one aspect, the described invention provides an implantcomprising

(a) a plurality of pieces comprising at least one tissue-derivedgrowth-conductive atrix; and

(b) at least one viable population of tissuegenic cells.

According to one embodiment, the at least one viable population oftissuegenic cells is adherent to and resident in an endogenous milieu ofthe growth-conductive matrix. According to another embodiment, the atleast one viable population of tissuegenic cells is caused to be incontact with the growth-conductive matrix.

According to one embodiment, the implant is an allogeneic implant.According to another embodiment, the implant is an autologous implant.According to another embodiment, the implant is a xenogeneic implant.

A Tissue Comprising a Matrix and Tissuegenic Cells

According to one embodiment, a tissue that comprises a growth-conductivematrix and at least one viable population of tissuegenic cells isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenumtissue, an epithelial tissue, a fascial tissue, a gastrointestinaltissue, a growth plate tissue, an intestinal mucosal tissue, anintestinal serosal tissue, an intervertebral disc tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a mammarytissue, a meniscal tissue, a muscle tissue, a nerve tissue, an ovarytissue, a pancreas tissue, a parenchymal organ tissue, a pericardialtissue, a periosteal tissue, a peritoneal tissue, a placental tissue, areproductive epithelial tissue, a respiratory epithelial tissue, a skintissue, a spleen tissue, a stomach tissue, a synovial tissue, a tendontissue, a testes tissue, an umbilical cord tissue, a urological tissue,a vascular tissue, a vein tissue, and, a combination thereof.

According to one embodiment, the tissue comprises an adipose tissue.According to one embodiment, the tissue comprises an amnion tissue.According to one embodiment, the tissue comprises an artery tissue.According to one embodiment, the tissue comprises a bone tissue.According to one embodiment, the tissue comprises a cartilage tissue.According to one embodiment, the tissue comprises a chorion tissue.According to one embodiment, the tissue comprises a colon tissue.According to one embodiment, the tissue comprises a dental tissue.According to one embodiment, the tissue comprises a dermal tissue.According to one embodiment, the tissue comprises a duodenal tissue.According to one embodiment, the tissue comprises an epithelial tissue.According to one embodiment, the tissue comprises a fascial tissue.According to one embodiment, the tissue comprises a gastrointestinaltissue. According to one embodiment, the tissue comprises a growth platetissue. According to one embodiment, the tissue comprises anintervertebral disc tissue. According to one embodiment, the tissuecomprises an intestinal mucosal tissue. According to one embodiment, thetissue comprises an intestinal serosal tissue. According to oneembodiment, the tissue comprises a kidney tissue. According to oneembodiment, the tissue comprises a ligament tissue. According to oneembodiment, the tissue comprises a liver tissue. According to oneembodiment, the tissue comprises a lung tissue. According to oneembodiment, the tissue comprises a mammary tissue. According to oneembodiment, the tissue comprises a meniscal tissue. According to oneembodiment, the tissue comprises a muscle tissue. According to oneembodiment, the tissue comprises a nerve tissue. According to oneembodiment, the tissue comprises an ovarian tissue. According to oneembodiment, the tissue comprises a pancreatic tissue. According to oneembodiment, the tissue comprises a parenchymal organ tissue. Accordingto one embodiment, the tissue comprises a pericardial tissue tissue.According to one embodiment, the tissue for fabricating the at implantcomprises a periosteal tissue. According to one embodiment, the tissuecomprises a peritoneal tissue. According to one embodiment, the tissuefor fabricating the at implant comprises a placental tissue. Accordingto one embodiment, the tissue for fabricating the at implant comprises areproductive epithelial tissue. According to one embodiment, the tissuefor fabricating the at implant comprises a respiratory epithelialtissue. According to one embodiment, the tissue comprises a skin tissue.According to one embodiment, the tissue comprises a spleen tissue.According to one embodiment, the tissue comprises a stomach tissue.According to one embodiment, the tissue comprises a synovial tissue.According to one embodiment, the implant comprises a tendon tissue.According to one embodiment, the tissue comprises a testes tissue.According to one embodiment, the tissue comprises an umbilical cordtissue. According to one embodiment, the implant comprises a tendontissue. According to one embodiment, the tissue comprises a urologicaltissue. According to one embodiment, the tissue comprises a vasculartissue. According to one embodiment, the tissue comprises a vein tissue.

According to one embodiment, the tissue is derived from a parenchymalorgan. The term “parenchymal organ” as used herein refers to an organ inwhich a specialized cell type carries out a specialized physiologicalfunction of the organ. According to one embodiment, the parenchymalorgan is selected from the group consisting of an artery, a brain, acolon, a duodenum, an intestinal mucosa, an intestinal serosa, a kidney,a liver, a lung, an ovary, a pancreas, a pericardium, a periotoneum, aspleen, a stomach, a testes, a vein, and a combination thereof.According to some embodiments, the parenchymal organ comprises anartery. According to some embodiments, the parenchymal organ comprises abrain. According to some embodiments, the parenchymal organ comprises acolon. According to some embodiments, the parenchymal organ comprises aduodenum. According to some embodiments, the parenchymal organ comprisesan intestinal mucosa. According to some embodiments, the parenchymalorgan comprises an intestinal serosa. According to some embodiments, theparenchymal organ comprises a kidney. According to some embodiments, theparenchymal organ comprises a liver. According to some embodiments, theparenchymal organ comprises a lung. According to some embodiments, theparenchymal organ comprises an ovary. According to some embodiments, theparenchymal organ comprises a pancreas. According to some embodiments,the parenchymal organ comprises a pericardium. According to someembodiments, the parenchymal organ comprises a periotoneum. According tosome embodiments, the parenchymal organ comprises a spleen. According tosome embodiments, the parenchymal organ comprises a stomach. Accordingto some embodiments, the parenchymal organ comprises a testes. Accordingto some embodiments, the parenchymal organ comprises a vein.

According to one embodiment, the tissue comprises an autologous tissue.According to another embodiment, the tissue comprises an allogeneictissue. According to another embodiment, the tissue comprises axenogeneic tissue.

According to one embodiment, the source of the tissue is a mammaliandonor. According to one embodiment, the source of the tissue is a humandonor. According to one embodiment, the human donor is a living donor.According to another embodiment, is the human donor is a cadevericdonor.

Adipose Tissue

According to some embodiments, the tissue comprises an adipose tissuederived from an adipose-rich body region.

According to some embodiments, the adipose rich body region is selectedfrom the group consisting of an abdomen, a hip, a hypodermal region ofskin, an infrapatellar fat pad, a knee, a mammary organ, a thigh, and, acombination thereof. According to some embodiments, the adipose richbody region is an abdomen. According to some embodiments, the adiposerich body region is a hip. According to some embodiments, the adiposerich body region is a hypodermal region of skin. According to someembodiments, the adipose rich body region is an infrapatellar fat pad.According to some embodiments, the adipose rich body region is a knee.According to some embodiments, the adipose rich body region is a mammaryorgan. According to some embodiments, the adipose rich body region is athigh.

According to some embodiments, the tissue is an adipose tissue selectedfrom the group consisting of a visceral adipose tissue, a subcutaneousadipose tissue and, a combination thereof. According to someembodiments, the tissue is an adipose tissue comprising a visceraladipose tissue. According to some embodiments, the tissue is an adiposetissue comprising a subcutaneous adipose tissue.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue is an adipose tissuederived from an adipose-rich body region of a human donor. According tosome embodiments, the human donor is a cadaveric donor. According tosome embodiments, the human donor is of a living donor.

According to one embodiment, the tissue is an adipose tissue derivedfrom an autologous adipose tissue. According to one embodiment, thetissue is an adipose tissue comprising an adipose tissue derived from anallogeneic adipose tissue. According to one embodiment, the tissue is anadipose tissue comprising an adipose tissue derived from a xenogeneicadipose tissue.

Bone Tissue

According to some embodiments, the tissue comprises a bone tissue or atleast one fragment thereof.

According to another embodiment, the bone tissue comprises a cancellousbone. According to some such embodiments, the cancellous bone isselected from the group consisting of a calcaneus, a distal femur bone,a proximal femur, a proximal humerus, an ilium, a patella, a distaltibia, a proximal tibia, a scapula, a cancellous bone from a sternum, atalus, at least one vertebral body and, a combination thereof. Accordingto some embodiments, the bone tissue comprises a periosteum.

According to some such embodiments, the cancellous bone comprisescancellous bone from a calcaneus. According to some such embodiments,the cancellous bone comprises cancellous bone from a distal femur.According to some such embodiments, the cancellous bone comprisescancellous bone from a proximal femur. According to some suchembodiments, the cancellous bone comprises cancellous bone from aproximal humerus. According to some such embodiments, the cancellousbone comprises cancellous bone from an ilium. According to some suchembodiments, the cancellous bone comprises cancellous bone from apatella. According to some such embodiments, the cancellous bonecomprises cancellous bone from a distal tibia. According to some suchembodiments, the cancellous bone comprises cancellous bone from aproximal tibia. According to some such embodiments, the cancellous bonecomprises cancellous bone from a scapula. According to some suchembodiments, the cancellous bone comprises cancellous bone from asternum. According to some such embodiments, the cancellous bonecomprises cancellous bone from a talus. According to some embodiments,the cancellous bone comprises cancellous bone from at least onevertebral body. A vertebral body refers to the largest portion of avertebral unit of a vertebral column.

According to another embodiment, the bone tissue comprises a corticalbone. According to some such embodiments, the cortical bone is selectedfrom the group consisting of a calcaneus, a distal femur bone, aproximal femur, a proximal humerus, an ilium, a patella, a distal tibia,a proximal tibia, a scapula, a cancellous bone from a sternum, a talus,at least one vertebral body and, a combination thereof.

According to some such embodiments, the cortical bone comprises corticalbone from a calcaneus. According to some such embodiments, the corticalbone comprises cortical bone from a distal femur. According to some suchembodiments, the cortical bone comprises cortical bone from a proximalfemur. According to some such embodiments, the cortical bone comprisescortical bone from a proximal humerus. According to some suchembodiments, the cortical bone comprises cortical bone from an ilium.According to some such embodiments, the cortical bone comprises corticalbone from a patella. According to some such embodiments, the corticalbone comprises cortical bone from a distal tibia. According to some suchembodiments, the cortical bone comprises cortical bone from a proximaltibia. According to some such embodiments, the cortical bone comprisescortical bone from a scapula. According to some such embodiments, thecortical bone comprises cortical bone from a sternum. According to somesuch embodiments, the cortical bone comprises cortical bone from atalus. According to some embodiments, the cortical bone comprisescortical bone from at least one vertebral body.

According to some such embodiments, the bone is at least one fragment ofan ilium. According to some such embodiments, the bone is at least onefragment of a long bone. According to some such embodiments, the longbone is selected from the group consisting of a femur, a fibula, ahumerus, a metacarpal, a metatarsal, a phalange, a radius, a tibia, anulna and, a combination thereof. According to some such embodiments, thelong bone is a femur. According to some such embodiments, the long boneis a fibula. According to some such embodiments, the long bone is ahumerus. According to some such embodiments, the long bone is ametacarpal. According to some such embodiments, the long bone is ametatarsal. According to some such embodiments, the long bone is aphalange. According to some such embodiments, the long bone is a radii.According to some such embodiments, the long bone is a tibia. Accordingto some such embodiments, the long bone is an ulna.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue is a bone tissuederived from a human donor. According to some embodiments human donor isa cadaveric donor. According to some embodiments, the human donor is aliving donor.

According to one embodiment, the tissue is a bone tissue derived from anautologous bone tissue. According to one embodiment, the tissue is abone tissue derived from an allogeneic bone tissue. According to oneembodiment, the tissue is a bone tissue derived from a xenogeneic bonetissue.

According to some embodiments, the synovial tissue comprises a synovialmembrane tissue. According to some embodiments, the synovial tissue isderived from a synovial joint. According to some such embodiments, thesynovial joint is at least one selected from the group consisting of aknee, an elbow, a shoulder, a hip, a condyloid joint, a pivot joint,and, a combination thereof. According to some such embodiments, thesynovial joint comprises a synovial joint of a knee. According to somesuch embodiments, the synovial joint comprises a synovial joint of anelbow. According to some such embodiments, the synovial joint comprisesa synovial joint of a shoulder. According to some such embodiments, thesynovial joint comprises a synovial joint of a hip. According to somesuch embodiments, the synovial joint comprises a condyloid joint.According to some such embodiments, the synovial joint comprises a pivotjoint.

According to some embodiments, the tissue comprises a tendon. As usedherein the term “tendon” refer to a nondistensible fibrous court or bandof variable length that is the part of the muscle that connects thefleshy/contractile part of muscle with its bony attachment or otherstructure. It consists of fascicles of very densely arranged, almostparallel collagenous fibers, rows of elongated fibrocytes, and a minimumof ground substance.

According to some embodiments, the tendon is derived from a cadavericdonor. According to some embodiments, the tendon is derived from aliving donor. According to one embodiment, the tissue is an autologoustendon. According to one embodiment, the tissue is an allogeneic tendon.According to one embodiment, the tissue is a xenogeneic tendon.

Cartilage Tissue

According to some embodiments, the tissue comprises a cartilage tissueselected from the group consisting of a hyaline cartilage tissue, afibrocartilage tissue, an elastic cartilage tissue and, a combinationthereof. According to some embodiments, the tissue comprises a hyalinecartilage tissue. According to some embodiments, the tissue comprises aa fibrocartilage cartilage tissue. According to some embodiments, thetissue comprises an elastic cartilage tissue.

According to some embodiments, the tissue comprises a cartilage tissuederived from a cartilaginous organ or at least one fragment thereof.

According to some embodiments, the cartilaginous organ is selected fromthe group consisting of an articular cartilage organ, a bronchus, agrowth plate, an intervertebral disc, a larynx, a meniscus, a nose, atrachea and, a combination thereof. According to some embodiments, thecartilaginous organ is an articular cartilage organ. According to someembodiments, the cartilaginous organ is a bronchus. According to someembodiments, the cartilaginous organ is a growth plate. According tosome embodiments, the cartilaginous organ is an intervertebral disc.According to some embodiments, the cartilaginous organ is a larynx.According to some embodiments, the cartilaginous organ is a meniscus.According to some embodiments, the cartilaginous organ is a nose.According to some embodiments, the cartilaginous organ is a trachea.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue comprises a cartilagetissue derived from a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, fromthe human donor is a living donor.

According to one embodiment, the tissue is a cartilage tissue derivedfrom an autologous cartilage tissue. According to one embodiment, thetissue is a cartilage tissue derived from an allogeneic cartilagetissue. According to one embodiment, the tissue is a cartilage tissuederived from a xenogeneic cartilage tissue.

Dental Tissue

According to some embodiments, the tissue comprises a dental tissue.According to some such embodiments, the tissue comprises a dental tissueselected from the group consisting of a tooth, a cementum tissue, adental pulp tissue, a dentin tissue, and an enamel tissue and, acombination thereof. According to some embodiments, the tissue comprisesa dental tissue comprising a cementum tissue. According to someembodiments, the tissue comprises a dental tissue comprising a dentalpulp tissue. According to some embodiments, the tissue comprises adental tissue comprising a dentin tissue. According to some embodiments,the tissue comprises a dental tissue comprising an enamel tissue.

According to some embodiments, the tissue comprises a dental tissuederived from at least one tooth or at least one fragment thereof.According to some embodiments, the tissue comprises a dental tissuederived from a plurality of teeth.

According to some embodiments, the tissue comprises a dental tissuederived from a tooth crown. According to some embodiments, the tissuecomprises a dental tissue derived from at least one fragment of a toothcrown. According to some embodiments, the tissue comprises a dentaltissue derived from a tooth root. According to some embodiments, thetissue comprises a dental tissue derived from at least one fragment of atooth root. According to some embodiments, the tissue comprises a dentaltissue derived from a tooth neck. According to some embodiments, thetissue is a dental tissue comprises a dental tissue derived from atleast one fragment of a tooth neck.

According to some embodiments, the tooth is selected from the groupconsisting of a deciduous tooth, a permanent tooth, and, a combinationthereof. According to some embodiments, the tooth is a deciduous tooth.According to some embodiments, the tooth is a permanent tooth.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue is a dental tissuederived from a human donor. According to some embodiments, the humandonor is a cadaveric donor. According to some embodiments, the humandonor is a living donor.

According to one embodiment, the tissue is an autologous dental tissue.According to one embodiment, the tissue is an allogeneic dental tissue.According to one embodiment, the tissue is a xenogeneic dental tissue.

Epithelial Tissue

According to some embodiments, the tissue comprises an epithelial tissueselected from the group consisting of a cutaneous epithelial tissue, amucuous epithelial tissue, a serous epithelial tissue and, a combinationthereof. According to some embodiments, the tissue comprises a cutaneousepithelial tissue. According to some embodiments, the tissue comprises amucuous epithelial tissue. According to some embodiments, the tissuecomprises a serous epithelial tissue. According to some embodiments, thetissue comprises a basement membrane tissue.

According to some embodiments, the tissue comprises an epithelial tissuederived from an epithelial organ or at least one fragment thereof.

According to some embodiments, the epithelial tissue is selected fromthe group consisting of a gastrointestinal lining, a pericardial lining,a peritoneal lining, a pleural lining, a reproductive, a respiratorylining, a urinary lining and, a combination thereof. According to someembodiments, the epithelial tissue is derived from a gastrointestinallining. According to one embodiment, the epithelial tissue is derivedfrom an intestinal mucosal lining. According to one embodiment, theepithelial tissue is derived from an intestinal serosal lining.According to some embodiments, the epithelial tissue is derived from apericardial lining. According to some embodiments, the epithelial tissueis derived from a peritoneal lining. According to some embodiments, theepithelial tissue is derived from a pleural lining. According to someembodiments, the epithelial tissue is derived from a reproductivelining. According to some embodiments, the epithelial tissue is derivedfrom a respiratory lining. According to some embodiments, the epithelialtissue is derived from a urinary lining.

According to some embodiments, the gastrointestinal lining is selectedfrom the group consisting of a duodenum lining, an esophagus lining, anileum lining, a jejunum lining, a large intestine lining, a mouthlining, a pharynx lining, a small intestine lining, a stomach liningand, a combination thereof. According to some embodiments, thegastrointestinal lining is a duodenum lining. According to someembodiments, the gastrointestinal lining is an esophagus lining.According to some embodiments, the gastrointestinal lining is an ileumlining. According to some embodiments, the gastrointestinal lining is ajejunum lining. According to some embodiments, the gastrointestinallining is a large intestine lining. According to some embodiments, thegastrointestinal lining is a pharynx lining. According to someembodiments, the gastrointestinal lining is a small intestine lining.According to some embodiments, the gastrointestinal lining is a stomachlining.

According to some embodiments, the epithelial organ is selected from thegroup consisting of a gastrointestinal organ, a respiratory organ, aurological organ and, a combination thereof. According to someembodiments, the epithelial organ comprises a gastrointestinal organ.According to some embodiments, the epithelial organ comprises arespiratory organ. According to some embodiments, the epithelial organcomprises a urological organ.

According to some embodiments, the gastrointestinal organ is selectedfrom the group consisting of a duodenum, an esophagus, an ileum, ajejunum, a large intestine, a mouth, a small intestine, a stomach and acombination thereof. According to some embodiments, the gastrointestinalorgan comprises a duodenum. According to some embodiments, thegastrointestinal organ comprises an esophagus. According to someembodiments, the gastrointestinal organ comprises an ileum. According tosome embodiments, the gastrointestinal organ comprises a jejunum.According to some embodiments, the gastrointestinal organ comprises alarge intestine. According to some embodiments, the gastrointestinalorgan comprises a small intestine. According to some embodiments, thegastrointestinal organ comprises a stomach.

According to some embodiments, the respiratory organ is selected fromthe group consisting of a bronchii, a diaphragm, a heart, a larynx, alung, a mouth, a nose, a pharynx, a trachea and a combination thereof.According to some embodiments, the respiratory organ comprises abronchii. According to some embodiments, the respiratory organ comprisesa diaphragm. According to some embodiments, the respiratory organcomprises a heart. According to some embodiments, the respiratory organcomprises a larynx. According to some embodiments, the respiratory organa lung. According to some embodiments, the respiratory organ comprises amouth. According to some embodiments, the respiratory organ comprises anose. According to some embodiments, the respiratory organ comprises apharynx. According to some embodiments, the respiratory organ comprisesa trachea.

According to some embodiments, the urological organ is selected from thegroup consisting of an adrenal gland, an epididymis, a kidney, an ovary,a penis, a prostate gland, a seminal vesicle, a testes, a ureter, aurethra, a urinary bladder, a vas deferens and a combination thereof.According to some embodiments, the urological organ comprises an adrenalgland. According to some embodiments, the urological organ comprises anepididymis. According to some embodiments, the urological organcomprises a kidney. According to some embodiments, the urological organcomprises an ovary. According to some embodiments, the urological organcomprises a penis. According to some embodiments, the urological organcomprises a prostate gland. According to some embodiments, theurological organ comprises a seminal vesicle. According to someembodiments, the urological organ comprises a testes. According to someembodiments, the urological organ comprises a ureter. According to someembodiments, the urological organ comprises a urethra. According to someembodiments, the urological organ comprises a urinary bladder. Accordingto some embodiments, the urological organ comprises a vas deferens.

According to some embodiments, the epithelial organ is selected from thegroup consisting of a duodenum, an esophagus, a heart, an ileum, ajejunum, a large intestine, a lung, a mouth, a pharynx, a smallintestine, a skin, a stomach, and, a combination thereof. According tosome embodiments, the epithelial organ comprises a duodenum. Accordingto some embodiments, the epithelial organ comprises an esophagus.According to some embodiments, the epithelial organ comprises a heart.According to some embodiments, the epithelial organ comprises an ileum.According to some embodiments, the epithelial organ comprises a jejunum.According to some embodiments, the epithelial organ comprises a largeintestine. According to some embodiments, the epithelial organ comprisesa lung. According to some embodiments, the epithelial organ comprises amouth. According to some embodiments, the epithelial organ comprises apharynx. According to some embodiments, the epithelial organ comprises asmall intestine. According to some embodiments, the epithelial organ iscomprises a skin. According to some embodiments, the epithelial organcomprises a stomach.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue is an epithelial tissuederived from a human donor. According to some embodiments, the humandonor is a cadaveric donor. According to some embodiments, human donoris a living donor.

According to one embodiment, the tissue is an epithelial tissue derivedfrom an autologous epithelial tissue. According to one embodiment, thetissue is an epithelial tissue derived from an allogeneic epithelialtissue. According to one embodiment, the tissue is an epithelial tissuederived from a xenogeneic epithelial tissue.

Fascial Tissue

According to some embodiments, the tissue comprises a fascial tissueselected from the group consisting of a superficial fascia, a deepfascia, a visceral fascia, and, a combination thereof. The term “fascia”as used herein refers to a fibroareolar connective tissue lamellaedistributed throughout the body surrounding delicate organs. Accordingto some embodiments, the tissue comprises a fascial tissue derived froma superficial fascia. According to some embodiments, the tissuecomprises a fascial tissue derived from a deep fascia. According to someembodiments, the tissue comprises a fascial tissue derived from avisceral fascia ligament.

According to some embodiments, the tissue comprises a fascial tissuederived from a fascia-rich body part or at least one fragment thereof.According to some embodiments, the fascia-rich body part is selectedfrom the group consisting of an arm, a back, an elbow, a foot, a hand, ahead, a knee, a leg, a muscle, a neck, a skin, a thigh, a toe, a wrist,and, a combination thereof. According to some embodiments, thefascia-rich body part comprises an arm. According to some embodiments,the fascia-rich body part comprises a back. According to someembodiments, the fascia-rich body part comprises an elbow. According tosome embodiments, the fascia-rich body part comprises a foot. Accordingto some embodiments, the fascia-rich body part comprises a hand.According to some embodiments, the fascia-rich body part comprises ahead. According to some embodiments, the fascia-rich body part comprisesa knee. According to some embodiments, the fascia-rich body partcomprises a leg. According to some embodiments, the fascia-rich bodypart comprises a muscle. According to some embodiments, the fascia-richbody part comprises a neck. According to some embodiments, thefascia-rich body part comprises a skin. According to some embodiments,the fascia-rich body part comprises a thigh. According to someembodiments, the fascia-rich body part comprises a toe. According tosome embodiments, the fascia-rich body part comprises a wrist.

According to some embodiments, the tissue comprises fascial tissueselected from the group consisting of a myofascia associated with amuscle, palmar fascia associated with a palm of a hand, plantar fasciaassociated with a sole of a foot, thoracolumbar fascia associated with aback, fascii lata associated with a thigh, tensor fascia lata associatedwith tendon tissue, and a combination thereof. According to someembodiments, the tissue comprises a fascial tissue derived frommyofascia associated with a muscle. According to some embodiments, thetissue comprises a fascial tissue derived from palmar fascia associatedwith a palm of a hand. According to some embodiments, the tissuecomprises a fascial tissue derived from plantar fascia associated with asole of a foot. According to some embodiments, the tissue comprises afascial tissue derived from thoracolumbar fascia associated with a back.According to some embodiments, the tissue comprises a fascial tissuederived from fascii lata associated with a thigh. According to someembodiments, the tissue comprises a fascial tissue derived from tensorfascia lata associated with tendon tissue.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue comprises a fasciatissue derived from a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, humandonor is a living donor.

According to one embodiment, the tissue is a fascial tissue derived froman autologous fascia. According to one embodiment, the tissue is afascial tissue derived from an allogeneic fascia. According to oneembodiment, the tissue is a fascial tissue derived from a xenogeneicfascia.

Ligament Tissue

According to some embodiments, the tissue comprises a ligament tissueselected from the group consisting of a capsular ligament, anextra-capsular ligament, an intracapsular ligament, a cruciate ligament,and, a combination thereof. The term “ligament” as used herein refers toa band or sheet of fibrous tissue connecting two or more bones,cartilages, or other structures, or serving as support for fasciae ormuscles and a fold of peritoneum supporting any of the abdominalviscera. According to some embodiments, the tissue comprises a ligamenttissue derived from a capsular ligament. According to some embodiments,the tissue comprises a ligament tissue derived from an extra-capsularligament. According to some embodiments, the tissue comprises a ligamenttissue derived from an intracapsular ligament. According to someembodiments, the tissue comprises a ligament tissue derived from acruciate ligament.

According to some embodiments, the tissue comprises a ligament tissuederived from a ligament-rich body part or at least one fragment thereof.According to some embodiments, the ligament-rich body part is selectedfrom the group consisting of an arm, an elbow, a foot, a hand, a head, aknee, a leg, a neck, a pelvis, a phalange, a thorax, a toe, a wrist,and, a combination thereof. According to some embodiments, theligament-rich body part comprises an arm. According to some embodiments,the ligament-rich body part comprises an elbow. According to someembodiments, the ligament-rich body part comprises a foot. According tosome embodiments, the ligament-rich body part comprises a hand.According to some embodiments, the ligament-rich body part comprises ahead. According to some embodiments, the ligament-rich body partcomprises a knee. According to some embodiments, the ligament-rich bodypart comprises a leg. According to some embodiments, the ligament-richbody part comprises a neck. According to some embodiments, theligament-rich body part comprises a pelvis. According to someembodiments, the ligament-rich body part comprises a phalange. Accordingto some embodiments, the ligament-rich body part comprises a thorax.According to some embodiments, the ligament-rich body part comprises atoe. According to some embodiments, the ligament-rich body partcomprises a wrist.

According to some embodiments, the tissue comprises a ligament tissuederived from a ligament organ or a fragment thereof. According to someembodiments, the ligament organ is selected from the group consisting ofa joint, a mouth, a patella, and, a combination thereof. According tosome embodiments, the ligament organ comprises a joint. According tosome embodiments, the ligament organ comprises a mouth. According tosome embodiments, the ligament organ comprises a patella.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue comprises a ligamenttissue derived from a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, humandonor is a living donor.

According to one embodiment, the tissue is a ligament tissue derivedfrom an autologous ligament. According to one embodiment, the tissue isa ligament tissue derived from an allogeneic ligament tissue. Accordingto one embodiment, the tissue is a ligament tissue derived from axenogeneic ligament tissue.

Mammary Tissue

According to some embodiments, the tissue comprises a mammary tissuederived from a mammary organ or at least one fragment thereof.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue is a mammary organ froma human donor. According to some embodiments, the human donor is acadaveric donor. According to some embodiments, the human donor is aliving donor.

According to one embodiment, the tissue is a mammary tissue derived froman autologous mammary organ. According to one embodiment, the tissue isa mammary tissue derived from an allogeneic mammary organ. According toone embodiment, the tissue is a mammary tissue derived from a xenogeneicmammary organ.

Muscle Tissue

According to some embodiments, the tissue comprises a muscle tissueselected from the group consisting of a cardiac muscle tissue, askeletal muscle tissue, a smooth muscle tissue, and, a combinationthereof. According to some embodiments, the tissue comprises a cardiacmuscle tissue. According to some embodiments, the tissue comprises askeletal muscle tissue. According to some embodiments, the tissuecomprises a smooth muscle tissue.

According to some embodiments, the tissue is a muscle tissue derivedfrom a muscle tissue-rich organ or at least one fragment thereof.

According to some embodiments, the muscle tissue-rich organ is selectedfrom the group consisting of a gastrointestinal organ, a skeletal organ,a heart, and, a combination thereof. According to some embodiments, themuscle tissue-rich organ comprises a gastrointestinal organ. Accordingto some embodiments, the muscle tissue-rich organ comprises a skeletalorgan. According to some embodiments, the muscle tissue-rich organcomprises a heart.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue comprises a muscletissue derived from a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, humandonor is a living donor.

According to one embodiment, the tissue comprises a muscle tissuederived from an autologous muscle tissue. According to one embodiment,the tissue comprises a muscle tissue derived from an allogeneic muscletissue. According to one embodiment, the tissue comprises a muscletissue derived from a xenogeneic muscle tissue.

Nerve Tissue

According to some embodiments, the tissue comprises a nerve tissuecomprising a nerve tissue derived from a nerve tissue-rich organ or atleast one fragment thereof.

According to some embodiments, the nerve tissue-rich organ is selectedfrom the group consisting of a brain, a spinal cord, and, a combinationthereof. According to some embodiments, the nerve tissue-rich organ is abrain. According to some embodiments, the nerve tissue-rich organ is aspinal cord.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the tissue is a nerve tissuederived from a human donor. According to some embodiments, the humandonor is a cadaveric donor.

According to one embodiment, the tissue comprises a nerve tissue derivedfrom an autologous nerve tissue. According to one embodiment, the tissuecomprises a nerve tissue derived from an allogeneic nerve tissue.According to one embodiment, the tissue comprises a nerve tissue derivedfrom a xenogeneic nerve tissue.

Placental Tissue

According to some embodiments, the tissue comprises a placental tissueselected from the group consisting of an amnion tissue, a choriontissue, an umbilical cord tissue, and, a combination thereof. Accordingto some embodiments, the tissue comprises an amnion tissue. According tosome embodiments, the tissue comprises a chorion tissue. According tosome embodiments, the tissue comprises an umbilical cord tissue.

According to some embodiments, the tissue comprises a placental tissuederived from an isolated placental organ or at least one fragmentthereof. According to some embodiments, the placental organ is selectedfrom the group consisting of an amnion, a chorion, an umbilical cord, aplacenta, and, a combination thereof. According to some embodiments, theplacental organ is an amnion. According to some embodiments, theplacental organ is a chorion. According to some embodiments, theplacental organ is a placenta. According to some embodiments, theplacental organ is an umbilical cord. According to some embodiments, thetissue comprises an umbilical cord tissue selected from the groupconsisting of an umbilical cord membrane tissue, umbilical cord blood,and, a combination thereof.

According to one embodiment, the tissue comprises a placental tissuederived from an autologous placental tissue. According to oneembodiment, the tissue comprises a placental tissue derived from anallogeneic placental tissue. According to one embodiment, the tissuecomprises a placental tissue derived from a xenogeneic placental tissue.

According to one embodiment, the tissue is an umbilical cord derivedfrom an autologous umbilical cord. According to one embodiment, thetissue is an umbilical cord tissue derived from an allogeneic umbilicalcord. According to one embodiment, the tissue is an umbilical cordtissue derived from a xenogeneic umbilical cord.

Skin Tissue

According to some embodiments, the tissue comprises a skin tissueselected from the group consisting of an epidermal tissue, a dermaltissue, a basement membrane tissue, and a combination thereof. Accordingto some embodiments, the skin tissue is an epidermal tissue. Accordingto some embodiments, the skin tissue is a dermal tissue. According tosome embodiments, the skin tissue is a basement membrane tissue.

According to one embodiment, the source of the tissue is a mammaliandonor. According to some embodiments, the skin tissue is derived from ahuman donor. According to some embodiments, the human donor is acadaveric donor. According to some embodiments, the human donor is aliving donor.

According to one embodiment, the tissue comprises a skin tissue derivedfrom an autologous skin tissue. According to one embodiment, the tissuecomprises a skin tissue derived from an allogeneic skin tissue.According to one embodiment, the tissue comprises a skin tissue derivedfrom a xenogeneic skin tissue.

Vascular Tissue

The term “vasculature” or “vascular tissue” as used herein refers to thevascular network of a part of the body and its arrangement. The vascularnetwork comprises blood vessels, i.e. any vessel conveying blood:arteries, arterioles, capillaries, venules, and veins. An artery is arelatively thick-walled, musclular pulsating vessel conveying blood awayfrom the heart. A vein is a blood vessel carrying blood toward theheart. Both arteries and veins comprises three layers: the tunicaintima, the tunica media and the tunica adventitia. Veins contain valvesthat prevent blood backflow. The tunica intima, a single layer of simplesquamous endothelial cells glued by a polysaccharide intercellularmatrix, surrounded by a thin layer of subendothelial connective tissueinterlaced with a number of circularly arranged elastic bands called theinternal elastic lamina; a tunica media, comprising circularly arrangedelastic fiber, connective tissue, polysaccharide substances, and a thickelastic band called the external elastic lamina, and the tunicaadventitia, entirely made of connective tissue. Capillaries comprise alayer of endothelium and connective tissue. According to someembodiments, the tissue comprises vascular tissue.

According to some embodiments, the vascular tissue is derived from acadaveric donor. According to some embodiments, the vascular tissue isderived from a living donor.

According to one embodiment, the tissue is a vascular tissue derivedfrom an autologous vascular tissue. According to one embodiment, thetissue is a vascular tissue derived from an allogeneic vascular tissue.According to one embodiment, the tissue is a vascular tissue derivedfrom a xenogeneic vascular tissue.

Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue is selected from the group consisting of anadipose tissue matrix, an amnion tissue matrix, an artery tissue matrix,a bone tissue matrix, a cartilage tissue matrix, a chorion tissuematrix, a colon tissue matrix, a dental tissue matrix, a dermal tissuematrix, a duodenum tissue matrix, an epithelial tissue matrix, a fascialtissue matrix, a gastrointestinal tissue matrix, a growth plate tissuematrix, an intervertebral disc tissue matrix, an intestinal mucosaltissue matrix, an s intestinal serosal tissue matrix, a kidney tissuematrix, a ligament tissue matrix, a liver tissue matrix, a lung tissuematrix, a mammary tissue matrix, a meniscal tissue matrix, a muscletissue matrix, a nerve tissue matrix, an ovary tissue, a pancreatictissue matrix, a parenchymal organ tissue matrix, a pericardial tissuematrix, a periosteal tissue matrix, a peritoneal tissue matrix, aplacental tissue matrix, a reproductive epithelial tissue matrix, arespiratory epithelial tissue matrix, a skin tissue matrix, a spleentissue matrix, a stomach tissue matrix, a synovial tissue matrix, atendon tissue matrix, a testes tissue matrix, an umbilical cord tissuematrix, a urological tissue matrix, a vascular tissue matrix, a veintissue matrix, and, a combination thereof.

According to one embodiment, the at least one growth-conductive matrixcomprises an adipose tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises an amnion tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises an artery tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a bone tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a cartilage tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a chorion tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a colon tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a dental tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a dermal tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a duodenal tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises an epithelial tissue matrix. According to one embodiment, theat least one growth-conductive matrix comprises a fascial tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a gastrointestinal tissue matrix. According to one embodiment,the at least one growth-conductive matrix comprises a growth platetissue matrix. According to one embodiment, the at least onegrowth-conductive matrix comprises an intervertebral disc tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises an intestinal mucosal tissue matrix. According to oneembodiment, the at least one growth-conductive matrix comprises anintestinal serosal tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a kidney tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a ligament tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a liver tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a lung tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a mammary tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a meniscal tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a muscle tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a nerve tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises an ovarian tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a pancreatic tissue matrix. According to one embodiment, theat least one growth-conductive matrix comprises a parenchymal organtissue matrix. According to one embodiment, the at least onegrowth-conductive matrix comprises a pericardial tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a periosteal tissue matrix. According to one embodiment, theat least one growth-conductive matrix comprises a peritoneal tissuematrix. According to one embodiment, the at least one growth-conductivematrix comprises a placental tissue matrix. According to one embodiment,the at least one growth-conductive matrix comprises a reproductiveepithelial tissue matrix. According to one embodiment, the at least onegrowth-conductive matrix comprises a respiratory epithelial tissuematrix. According to one embodiment, the at least one growth-conductivematrix comprises a skin tissue matrix. According to one embodiment, theat least one growth-conductive matrix comprises a spleen tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a stomach tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a synovial tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises a tendon tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises a testes tissue matrix.According to one embodiment, the at least one growth-conductive matrixcomprises an umbilical cord tissue matrix. According to one embodiment,the at least one growth-conductive matrix comprises a urological tissuematrix. According to one embodiment, the at least one growth-conductivematrix comprises a vascular tissue matrix. According to one embodiment,the at least one growth-conductive matrix comprises a vein tissuematrix.

According to one embodiment, the at least one growth-conductive matrixcomprises a purified collagen matrix. According to one embodiment, theat least one growth-conductive matrix comprises a calcium phosphateceramic matrix. According to one embodiment, the at least onegrowth-conductive matrix comprises a calcium phosphate ceramic matrix.

According to one embodiment, the at least one growth-conductive matrixis derived from an autologous tissue. According to one embodiment, theat least one growth-conductive matrix is derived from an allogeneictissue. According to one embodiment, the at least one growth-conductivematrix is derived from a xenogeneic tissue.

According to one embodiment, the source of the tissue is a mammaliandonor. According to one embodiment, the at least one growth-conductivematrix is derived from a human donor. According to one embodiment, theat least one growth-conductive matrix is derived from a living donor.According to one embodiment, the at least one growth-conductive matrixis derived from a cadeveric donor.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a tissue-derived growth conductivematrix from which unwanted cells have been removed.

Adipose Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixcomprises an adipose tissue matrix. According to one embodiment, the atleast one growth-conductive matrix comprises an adipose tissue matrixderived from an adipose-rich body region.

According to some embodiments, the adipose rich body region is selectedfrom the group consisting of an abdomen, a hip, a hypodermal region ofskin, an infrapatellar fat pad, a knee, a mammary organ, a thigh and, acombination thereof. According to some embodiments, the adipose richbody region is an abdomen. According to some embodiments, the adiposerich body region is a hip. According to some embodiments, the adiposerich body region is a hypodermal region of skin. According to someembodiments, the adipose rich body region is an infrapatellar fat pad.According to some embodiments, the adipose rich body region is a knee.According to some embodiments, the adipose rich body region is a mammaryorgan. According to some embodiments, the adipose rich body region is athigh.

According to some embodiments, the adipose tissue matrix comprisestissue matrix derived from an adipose tissue selected from the groupconsisting of a visceral adipose tissue, a subcutaneous adipose tissueand, a combination thereof. According to some embodiments, the adiposetissue matrix comprises tissue matrix derived from a visceral adiposetissue. According to some embodiments, the adipose tissue matrixcomprises tissue matrix derived from a subcutaneous adipose tissue.

According to some embodiments, the adipose tissue matrix comprisestissue matrix derived from an adipose-rich body region of a human donor.According to some embodiments, the human donor is a cadaveric donor.According to some embodiments, the human donor is a living donor.

According to one embodiment, the adipose tissue matrix comprises tissuematrix derived from an autologous adipose tissue matrix. According toone embodiment, the adipose tissue matrix comprises tissue matrixderived from an allogeneic adipose tissue matrix. According to oneembodiment, the adipose tissue matrix comprises tissue matrix derivedfrom a xenogeneic adipose tissue matrix.

According to one embodiment, the at least one adipose tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one adipose tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises an adipose tissue-derived growthconductive matrix from which unwanted cells have been removed.

Bone Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixcomprises a bone tissue matrix. According to some embodiments, the bonetissue matrix is derived from a bone or at least one fragment thereof.

According to another embodiment, the bone comprises a cancellous bone.According to some such embodiments, the cancellous bone is selected fromthe group consisting of a cancellous bone from a calcaneus, a cancellousbone from a distal femur, a cancellous bone from a proximal femur, acancellous bone from a proximal humerus, a cancellous bone from a ilium,a cancellous bone from a patella, a cancellous bone from a distal tibia,a cancellous bone from a proximal tibia, a cancellous bone from ascapula, a cancellous bone from a sternum, a cancellous bone from atalus, a cancellous bone from at least one vertebral body and, acombination thereof. According to some embodiments, the bone tissuecomprises a periosteum.

According to some such embodiments, the cancellous bone comprisescancellous bone from a calcaneus. According to some such embodiments,the cancellous bone comprises cancellous bone from a distal femur.According to some such embodiments, the cancellous bone comprisescancellous bone from a proximal femur. According to some suchembodiments, the cancellous bone comprises cancellous bone from aproximal humerus. According to some such embodiments, the cancellousbone comprises cancellous bone from an ilium. According to some suchembodiments, the cancellous bone comprises cancellous bone from apatella. According to some such embodiments, the cancellous bonecomprises cancellous bone from a distal tibia. According to some suchembodiments, the cancellous bone comprises cancellous bone from aproximal tibia. According to some such embodiments, the cancellous bonecomprises cancellous bone from a scapula. According to some suchembodiments, the cancellous bone comprises cancellous bone from asternum. According to some such embodiments, the cancellous bonecomprises cancellous bone from a talus. According to some embodiments,the cancellous bone comprises cancellous bone from at least onevertebral body.

According to another embodiment, the bone comprises cortical bone.According to some such embodiments, the cortical bone is selected fromthe group consisting of a calcaneus, a distal femur bone, a proximalfemur, a proximal humerus, an ilium, a patella, a distal tibia, aproximal tibia, a scapula, a cancellous bone from a sternum, a talus, atleast one vertebral body and, a combination thereof.

According to some such embodiments, the cortical bone comprises corticalbone from a calcaneus. According to some such embodiments, the corticalbone comprises cortical bone from a distal femur. According to some suchembodiments, the cortical bone comprises cortical bone from a proximalfemur. According to some such embodiments, the cortical bone comprisescortical bone from a proximal humerus. According to some suchembodiments, the cortical bone comprises cortical bone from an ilium.According to some such embodiments, the cortical bone comprises corticalbone from a patella. According to some such embodiments, the corticalbone comprises cortical bone from a distal tibia. According to some suchembodiments, the cortical bone comprises cortical bone from a proximaltibia. According to some such embodiments, the cortical bone comprisescortical bone from a scapula. According to some such embodiments, thecortical bone comprises cortical bone from a sternum. According to somesuch embodiments, the cortical bone comprises cortical bone from atalus. According to some embodiments, the cortical bone comprisescortical bone from at least one vertebral body.

According to some such embodiments, the bone is at least one fragment ofan ilium. According to some such embodiments, the bone is at least onefragment of a long bone. According to some such embodiments, the longbone is selected from the group consisting of a femur, a fibula, ahumerus, a metacarpal, a metatarsal, a phalange, a radii, a tibia, anulna and, a combination thereof. According to some such embodiments, thelong bone is a femur. According to some such embodiments, the long boneis a fibula. According to some such embodiments, the long bone is ahumerus. According to some such embodiments, the long bone is ametacarpal. According to some such embodiments, the long bone is ametatarsal. According to some such embodiments, the long bone is aphalange. According to some such embodiments, the long bone is a radii.According to some such embodiments, the long bone is a tibia. Accordingto some such embodiments, the long bone is an ulna.

According to another embodiment, the at least one growth-conductivematrix comprises cancellous bone and cortical bone. According to someembodiments, the at least one growth-conductive matrix comprisescancellous bone and cortical bone in a ratio of about 60:40 w/w.According to some embodiments, the at least one growth-conductive matrixcomprises cancellous bone and cortical bone in a ratio of about 65:35w/w. According to some embodiments, the at least one growth-conductivematrix comprises cancellous bone and cortical bone in a ratio of about66:34 w/w. According to some embodiments, the at least onegrowth-conductive matrix comprises cancellous bone and cortical bone ina ratio of about 67:33 w/w. According to some embodiments, the at leastone growth-conductive matrix comprises cancellous bone and cortical bonein a ratio of about 68:32 w/w. According to some embodiments, the atleast one growth-conductive matrix comprises cancellous bone andcortical bone in a ratio of about 69:31 w/w. According to someembodiments, the at least one growth-conductive matrix comprisescancellous bone and cortical bone in a ratio of about 70:30 w/w.According to some embodiments, the at least one growth-conductive matrixcomprises cancellous bone and cortical bone in a ratio of about 75:25w/w. According to some embodiments, the at least one growth-conductivematrix comprises cancellous bone and cortical bone in a ratio of about80:20 w/w. According to some embodiments, the at least onegrowth-conductive matrix comprises cancellous bone and cortical bone ina ratio of about 85:15 w/w. According to some embodiments, the at leastone growth-conductive matrix comprises cancellous bone and cortical bonein a ratio of about 90:10 w/w. According to some embodiments, the atleast one growth-conductive matrix comprises cancellous bone andcortical bone in a ratio of about 95:5 w/w. According to someembodiments, the at least one growth-conductive matrix comprisescancellous bone and cortical bone in a ratio of about 96:4 w/w.According to some embodiments, the at least one growth-conductive matrixcomprises cancellous bone and cortical bone in a ratio of about 97:3w/w. According to some embodiments, the at least one growth-conductivematrix comprises cancellous bone and cortical bone in a ratio of about98:2 w/w. According to some embodiments, the at least onegrowth-conductive matrix comprises cancellous bone and cortical bone ina ratio of about 99:1 w/w.

According to one embodiment, the at least one growth-conductive matrixderived from a bone tissue matrix comprises an osteoconductive matrix.According to one embodiment, the at least one osteoconductive matrix isbone. According to another embodiment, the at least one osteoconductivematrix is at least one fragment of bone.

According to another embodiment, the at least one osteoconductive matrixcomprises cancellous bone. According to some such embodiments, thecancellous bone is cancellous bone from a proximal femur. According tosome such embodiments, the cancellous bone is cancellous bone from adistal femur. According to some such embodiments, the cancellous bone iscancellous bone from a proximal tibia. According to some suchembodiments, the cancellous bone is cancellous bone from a distal tibia.According to some such embodiments, the cancellous bone is cancellousbone from a patella. According to some such embodiments, the cancellousbone is cancellous bone from a calcaneus. According to some suchembodiments, the cancellous bone is cancellous bone from a talus.According to some such embodiments, the cancellous bone is cancellousbone from a proximal humerus. According to some such embodiments, thecancellous bone is cancellous bone from a scapula. According to somesuch embodiments, the cancellous bone is cancellous bone from a sternum.According to some such embodiments, the cancellous bone is cancellousbone from an ilium. According to some embodiments, the at least oneosteoconductive comprises cancellous bone from at least one vertebralbody.

According to another embodiment, the at least one osteoconductive matrixcomprises cortical bone. According to some such embodiments, thecortical bone is cortical bone from a proximal femur. According to somesuch embodiments, the cortical bone is cortical bone from a distalfemur. According to some such embodiments, the cortical bone is corticalbone from a proximal tibia. According to some such embodiments, thecortical bone is cortical bone from a distal tibia. According to somesuch embodiments, the cortical bone is cortical bone from a patella.According to some such embodiments, the cortical bone is cortical bonefrom a calcaneus. According to some such embodiments, the cortical boneis cortical bone from a talus. According to some such embodiments, thecortical bone is cortical bone from a proximal humerus. According tosome such embodiments, the cortical bone is cortical bone from ascapula. According to some such embodiments, the cortical bone iscortical bone from a sternum. According to some such embodiments, thecortical bone is cortical bone from an ilium. According to someembodiments, the at least one osteoconductive matrix comprises corticalbone from at least one vertebral body.

According to another embodiment, the at least one osteoconductive matrixcomprises cancellous bone and cortical bone. According to someembodiments, the at least one osteoconductive matrix comprisescancellous bone and cortical bone in a ratio of about 60:40 w/w.According to some embodiments, the at least one osteoconductive matrixcomprises cancellous bone and cortical bone in a ratio of about 65:35w/w. According to some embodiments, the at least one growth-conductivematrix comprises cancellous bone and cortical bone in a ratio of about66:34 w/w. According to some embodiments, the at least onegrowth-conductive matrix comprises cancellous bone and cortical bone ina ratio of about 67:33 w/w. According to some embodiments, the at leastone growth-conductive matrix comprises cancellous bone and cortical bonein a ratio of about 68:32 w/w. According to some embodiments, the atleast one growth-conductive matrix comprises cancellous bone andcortical bone in a ratio of about 69:31 w/w. According to someembodiments, the at least one osteoconductive matrix comprisescancellous bone and cortical bone in a ratio of about 70:30 w/w.According to some embodiments, the at least one osteoconductive matrixcomprises cancellous bone and cortical bone in a ratio of about 75:25w/w. According to some embodiments, the at least one osteoconductivematrix comprises cancellous bone and cortical bone in a ratio of about80:20 w/w. According to some embodiments, the at least oneosteoconductive matrix comprises cancellous bone and cortical bone in aratio of about 85:15 w/w. According to some embodiments, the at leastone osteoconductive matrix comprises cancellous bone and cortical bonein a ratio of about 90:10 w/w. According to some embodiments, the atleast one osteoconductive matrix comprises cancellous bone and corticalbone in a ratio of about 95:5 w/w. According to some embodiments, the atleast one osteoconductive matrix comprises cancellous bone and corticalbone in a ratio of about 96:4 w/w. According to some embodiments, the atleast one osteoconductive matrix comprises cancellous bone and corticalbone in a ratio of about 97:3 w/w. According to some embodiments, the atleast one osteoconductive matrix comprises cancellous bone and corticalbone in a ratio of about 98:2 w/w. According to some embodiments, the atleast one osteoconductive matrix comprises cancellous bone and corticalbone in a ratio of about 99:1 w/w.

According to some embodiments, the bone tissue matrix comprises tissuematrix derived from a bone matrix from a human donor. According to someembodiments, the human donor is a cadaveric donor. According to someembodiments, the human donor is a living donor.

According to some embodiments, the bone tissue matrix is derived from anautologous bone tissue. According to some embodiments, the bone tissuematrix is derived from an allogeneic bone tissue. According to someembodiments, the bone tissue matrix is derived from a xenogeneic bonetissue.

According to one embodiment, the at least one bone tissue-derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one growth-conductive matrix.

According to one embodiment, the at least one growth-conductive matrixcomprises a synovial tissue matrix. According to some embodiments, thesynovial tissue comprises a synovial membrane. According to someembodiments, the synovial tissue is derived from a synovial joint.According to some such embodiments, the synovial joint is at least oneselected from the group consisting of a knee, an elbow, a shoulder, ahip, a condyloid joint, a pivot joint, and, a combination thereof.According to some such embodiments, the synovial joint comprises asynovial joint of a knee. According to some such embodiments, thesynovial joint comprises a synovial joint of an elbow. According to somesuch embodiments, the synovial joint comprises a synovial joint of ashoulder. According to some such embodiments, the synovial jointcomprises a synovial joint of a hip. According to some such embodiments,the synovial joint comprises a condyloid joint. According to some suchembodiments, the synovial joint comprises a pivot joint.

According to one embodiment, the at least one growth-conductive matrixcomprises a tendon tissue matrix. According to some embodiments, thetendon tissue matrix comprises tissue matrix derived from a tendon. Asused herein the term “tendon” refer to a nondistensible fibrous court orband of variable length that is the part of the muscle that connects thefleshy/contractile part of muscle with its bony attachment or otherstructure. It consists of fascicles of very densely arranged, almostparallel collagenous fibers, rows of elongated fibrocytes, and a minimumof ground substance.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a bone tissue-derived growthconductive matrix from which unwanted cells have been removed. Accordingto one embodiment, the at least one tissue derived growth-conductivematrix comprises a periosteal tissue-derived growth conductive matrixfrom which unwanted cells have been removed. According to oneembodiment, the at least one tissue derived growth-conductive matrixcomprises a synovial tissue-derived growth conductive matrix from whichunwanted cells have been removed.

Cartilage Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a cartilage tissue matrix. According tosome embodiments, the cartilage tissue matrix is derived from acartilage tissue selected from the group consisting of a hyalinecartilage, a fibrocartilage tissue, an elastic cartilage tissue and, acombination thereof. According to some embodiments, the cartilage tissuematrix is derived from a hyaline cartilage tissue. According to someembodiments, the cartilage tissue matrix is derived from afibrocartilage cartilage tissue. According to some embodiments, thecartilage tissue matrix is derived from an elastic cartilage tissue.

According to some embodiments, the cartilage tissue matrix is derivedfrom a cartilaginous organ or at least one fragment thereof.

According to some embodiments, the cartilaginous organ is selected fromthe group consisting of an articular cartilage organ, a bronchus, agrowth plate, an intervertebral disc, a larynx, a meniscus, a nose, atrachea and, a combination thereof. According to some embodiments, thecartilaginous organ comprises an articular cartilage organ. According tosome embodiments, the cartilaginous organ comprises a bronchus.According to some embodiments, the cartilaginous organ comprises agrowth plate. According to some embodiments, the cartilaginous organcomprises an intervertebral disc. According to some embodiments, thecartilaginous organ comprises a larynx. According to some embodiments,the cartilaginous organ comprises a meniscus. According to someembodiments, the cartilaginous organ comprises a nose. According to someembodiments, the cartilaginous organ comprises a trachea.

According to some embodiments, the cartilage tissue matrix is derivedfrom a cartilage tissue from a human donor. According to someembodiments, the human donor is a cadaveric donor. According to someembodiments, the human donor is a living donor.

According to one embodiment, the cartilage tissue matrix is derived froman autologous cartilage tissue. According to one embodiment, thecartilage tissue matrix is derived from an allogeneic cartilage tissue.According to one embodiment, the cartilage tissue matrix is derived froma xenogeneic cartilage tissue.

According to one embodiment, the at least one cartilage tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one cartilage tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a cartilage tissue-derived growthconductive matrix from which unwanted cells have been removed. Accordingto one embodiment, the at least one tissue derived growth-conductivematrix comprises a bronchial tissue-derived growth conductive matrixfrom which unwanted cells have been removed. According to oneembodiment, the at least one tissue derived growth-conductive matrixcomprises a growth plate tissue-derived growth conductive matrix fromwhich unwanted cells have been removed. According to one embodiment, theat least one tissue derived growth-conductive matrix comprises aninvertebral plate tissue-derived growth conductive matrix from whichunwanted cells have been removed. According to one embodiment, the atleast one tissue derived growth-conductive matrix comprises a larynxtissue-derived growth conductive matrix from which unwanted cells havebeen removed. According to one embodiment, the at least one tissuederived growth-conductive matrix comprises a meniscal tissue-derivedgrowth conductive matrix from which unwanted cells have been removed.According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a tracheal tissue-derived growthconductive matrix from which unwanted cells have been removed.

Dental Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a dental tissue matrix. According tosome embodiments, the dental tissue matrix is derived from a dentaltissue selected from the group consisting of a cementum tissue, a dentalpulp tissue, a dentin tissue, an enamel tissue and, a combinationthereof. According to some embodiments, the at least onegrowth-conductive matrix is derived from a cementum tissue. According tosome embodiments the at least one growth-conductive matrix is derivedfrom a dental pulp tissue. According to some embodiments, the at leastone growth-conductive matrix is derived from a dentin tissue. Accordingto some embodiments, the at least one growth-conductive matrix isderived from an enamel tissue.

According to some embodiments, the dental tissue matrix comprises tissuematrix derived from at least one tooth or at least one fragment thereof.

According to some embodiments, the dental tissue matrix is derived froma tooth crown or at least one fragment therefrom. According to someembodiments, the dental tissue matrix is derived from a tooth root or atleast one fragment therefrom. According to some embodiments, the dentaltissue matrix is derived from a tooth neck or at least one fragmenttherefrom.

According to some embodiments, the tooth is selected from the groupconsisting of a deciduous tooth, a permanent tooth and a combinationtherefrom. According to some embodiments, the tooth is a deciduoustooth. According to some embodiments, tooth is a permanent tooth.

According to some embodiments, the dental tissue matrix is derived froma dental tissue from a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, thehuman donor is a living donor.

According to one embodiment, the dental tissue matrix is derived from anautologous dental tissue. According to one embodiment, the dental tissuematrix is derived from an allogeneic dental tissue. According to oneembodiment, the dental tissue matrix is derived from a xenogeneic dentaltissue.

According to one embodiment, the at least one dental tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one dental tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a dental tissue-derived growthconductive matrix from which unwanted cells have been removed.

Epithelial Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises an epithelial tissue matrix. Accordingto some embodiments, the epithelial tissue matrix is derived from anepithelial tissue selected from the group consisting of a cutaneousepithelial tissue, a mucuous epithelial tissue, a serous epithelialtissue and, a combination thereof. According to some embodiments, theepithelial tissue matrix is derived from a cutaneous epithelial tissue.According to some embodiments, the epithelial tissue matrix is derivedfrom a mucuous epithelial tissue. According to some embodiments, theepithelial tissue matrix is derived from a serous epithelial tissue.According to some embodiments, the epithelial tissue matrix is derivedfrom a basement membrane tissue. According to one embodiment, theepithelial tissue matrix is derived from an intestinal mucosal lining.According to one embodiment, the epithelial tissue matrix is derivedfrom an intestinal serosal lining.

According to some embodiments, the epithelial tissue is selected fromthe group consisting of a gastrointestinal lining, a pericardial lining,a peritoneal lining, a pleural lining, a reproductive lining, arespiratory lining, a urinary lining and, a combination thereof.According to some embodiments, the epithelial tissue is derived from agastrointestinal lining. According to some embodiments, the epithelialtissue is derived from a pericardial lining. According to someembodiments, the epithelial tissue is derived from a peritoneal lining.According to some embodiments, the epithelial tissue is derived from apleural lining. According to some embodiments, the epithelial tissue isderived from a reproductive lining. According to some embodiments, theepithelial tissue is derived from a respiratory lining. According tosome embodiments, the epithelial tissue is derived from a urinarylining.

According to some embodiments, the gastrointestinal lining is selectedfrom the group consisting of a duodenum lining, an esophagus lining, anileum lining, a jejunum lining, a large intestine lining, a mouthlining, a pharynx lining, a small intestine lining, a stomach liningand, a combination thereof. According to some embodiments, thegastrointestinal lining is a duodenum lining. According to someembodiments, the gastrointestinal lining is an esophagus lining.According to some embodiments, the gastrointestinal lining is an ileumlining. According to some embodiments, the gastrointestinal lining is ajejunum lining. According to some embodiments, the gastrointestinallining is a large intestine lining. According to some embodiments, thegastrointestinal lining is a pharynx lining. According to someembodiments, the gastrointestinal lining is a small intestine lining.According to some embodiments, the gastrointestinal lining is a stomachlining.

According to some embodiments, the epithelial tissue matrix is derivedfrom an epithelial organ or at least one fragment thereof.

According to some embodiments, the epithelial organ is selected from thegroup consisting of a gastrointestinal organ, a respiratory organ, aurological organ and, a combination thereof. According to someembodiments, the epithelial organ comprises a gastrointestinal organ.According to some embodiments, the epithelial organ comprises arespiratory organ. According to some embodiments, the epithelial organcomprises a urological organ.

According to some embodiments, the gastrointestinal organ is selectedfrom the group consisting of a duodenum, an esophagus, an ileum, ajejunum, a large intestine, a mouth, a small intestine, a stomach and acombination thereof. According to some embodiments, the gastrointestinalorgan comprises a duodenum. According to some embodiments, thegastrointestinal organ comprises an esophagus. According to someembodiments, the gastrointestinal organ comprises an ileum. According tosome embodiments, the gastrointestinal organ comprises a jejunum.According to some embodiments, the gastrointestinal organ comprises alarge intestine. According to some embodiments, the gastrointestinalorgan comprises a small intestine. According to some embodiments, thegastrointestinal organ comprises a stomach.

According to some embodiments, the respiratory organ is selected fromthe group consisting of a bronchii, a diaphragm, a heart, a larynx, alung, a mouth, a nose, a pharynx, a trachea and a combination thereof.According to some embodiments, the respiratory organ comprises abronchii. According to some embodiments, the respiratory organ comprisesa diaphragm. According to some embodiments, the respiratory organcomprises a heart. According to some embodiments, the respiratory organcomprises a larynx. According to some embodiments, the respiratory organa lung. According to some embodiments, the respiratory organ comprises amouth. According to some embodiments, the respiratory organ comprises anose. According to some embodiments, the respiratory organ comprises apharynx. According to some embodiments, the respiratory organ comprisesa trachea.

According to some embodiments, the urological organ is selected from thegroup consisting of an adrenal gland, an epididymis, a kidney, an ovary,a penis, a prostate, a seminal vesicle, a testes, a ureter, a urethra, aurinary bladder, a vas deferens and a combination thereof. According tosome embodiments, the urological organ comprises an adrenal gland.According to some embodiments, the urological organ comprises anepididymis. According to some embodiments, the urological organcomprises a kidney. According to some embodiments, the urological organcomprises an ovary. According to some embodiments, the urological organcomprises a penis. According to some embodiments, the urological organcomprises a prostate. According to some embodiments, the urologicalorgan comprises a seminal vesicle. According to some embodiments, theurological organ comprises a testes. According to some embodiments, theurological organ comprises a ureter. According to some embodiments, theurological organ comprises a urethra. According to some embodiments, theurological organ comprises a urinary bladder. According to someembodiments, the urological organ comprises a vas deferens.

According to some embodiments, the epithelial organ is selected from thegroup consisting of a duodenum, an esophagus, a heart, an ileum, ajejunum, a large intestine, a lung, a mouth, a pharynx, a smallintestine, a skin, a stomach and, a combination thereof. According tosome embodiments, the epithelial organ comprises a duodenum. Accordingto some embodiments, the epithelial organ comprises an esophagus.According to some embodiments, the epithelial organ comprises a heart.According to some embodiments, the epithelial organ comprises an ileum.According to some embodiments, the epithelial organ comprises a jejunum.According to some embodiments, the epithelial organ comprises a largeintestine. According to some embodiments, the epithelial organ comprisesa lung. According to some embodiments, the epithelial organ comprises amouth. According to some embodiments, the epithelial organ comprises apharynx. According to some embodiments, the epithelial organ comprises asmall intestine. According to some embodiments, the epithelial organcomprises a skin. According to some embodiments, the epithelial organcomprises a stomach.

According to some embodiments, the epithelial tissue matrix is derivedfrom an epithelial tissue from a human donor. According to someembodiments, the human donor is a cadaveric donor. According to someembodiments, the human donor is a living donor.

According to one embodiment, the epithelial tissue matrix is derivedfrom an autologous epithelial tissue. According to one embodiment, theepithelial tissue matrix is derived from an allogeneic epithelialtissue. According to one embodiment, the epithelial tissue matrix isderived from a xenogeneic epithelial tissue.

According to one embodiment, the at least one epithelial tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one epithelial tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises an epithelial tissue-derived growthconductive matrix from which unwanted cells have been removed. Accordingto one embodiment, the at least one tissue derived growth-conductivematrix comprises a gastrointestinal tissue-derived growth conductivematrix from which unwanted cells have been removed. According to oneembodiment, the at least one tissue derived growth-conductive matrixcomprises a liver tissue-derived growth conductive matrix from whichunwanted cells have been removed. According to one embodiment, the atleast one tissue derived growth-conductive matrix comprises a lungtissue-derived growth conductive matrix from which unwanted cells havebeen removed. According to one embodiment, the at least one tissuederived growth-conductive matrix comprises a urological tissue-derivedgrowth conductive matrix from which unwanted cells have been removed.

Fascial Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a fascial tissue matrix. According tosome embodiments, the fascial tissue matrix is derived from a fascialtissue selected from the group consisting of a superficial fascia, adeep fascia, a visceral fascia, and, a combination thereof. The term“fascia” as used herein refers to a fibroareolar connective tissuelamellae distributed throughout the body surrounding delicate organs.According to some embodiments, the fascial tissue matrix is derived froma superficial fascia. According to some embodiments, the fascial tissuematrix is derived from a deep fascia. According to some embodiments, thefascial tissue matrix is a visceral fascia.

According to some embodiments, the fascial tissue matrix is derived froma fascia-rich body part or at least one fragment thereof. According tosome embodiments, the fascia-rich body part is selected from the groupconsisting of an arm, a back, an elbow, a foot, a hand, a head, a knee,a leg, a muscle, a neck, a skin, a thigh, a toe, a wrist, and, acombination thereof. According to some embodiments, the fascia-rich bodypart comprises an arm. According to some embodiments, the fascia-richbody part comprises a back. According to some embodiments, thefascia-rich body part comprises an elbow. According to some embodiments,the fascia-rich body part comprises a foot. According to someembodiments, the fascia-rich body part comprises a hand. According tosome embodiments, the fascia-rich body part comprises a head. Accordingto some embodiments, the fascia-rich body part comprises a knee.According to some embodiments, the fascia-rich body part comprises aleg. According to some embodiments, the fascia-rich body part comprisesa muscle. According to some embodiments, the fascia-rich body partcomprises a neck. According to some embodiments, the fascia-rich bodypart comprises a skin. According to some embodiments, the fascia-richbody part comprises a thigh. According to some embodiments, thefascia-rich body part comprises a toe. According to some embodiments,the fascia-rich body part comprises a wrist.

According to some embodiments, the fascial tissue matrix is derived froma fascial tissue selected from the group consisting of a myofasciaassociated with a muscle, palmar fascia associated with a palm of ahand, plantar fascia associated with a sole of a foot, thoracolumbarfascia associated with a back, fascii lata associated with a thigh,tensor fascia lata associated with tendon tissue, and a combinationthereof. According to some embodiments, the fascial tissue matrix isderived from myofascia associated with a muscle. According to someembodiments, fascial tissue matrix is derived from palmar fasciaassociated with a palm of a hand. According to some embodiments, fascialtissue matrix is derived from plantar fascia associated with a sole of afoot. According to some embodiments, fascial tissue matrix is derivedfrom thoracolumbar fascia associated with a back. According to someembodiments, fascial tissue matrix is derived from fascii lataassociated with a thigh. According to some embodiments, fascial tissuematrix is derived from tensor fascia lata associated with tendon tissue.

According to some embodiments, the fascial tissue matrix is derived froma fascial tissue from a mammalian donor. According to some embodiments,the fascial tissue matrix is derived from a fascial tissue from a humandonor. According to some embodiments, the human donor is a cadavericdonor. According to some embodiments, the human donor is a living donor.

According to one embodiment, the fascial tissue matrix is derived froman autologous fascial tissue. According to one embodiment, the fascialtissue matrix is derived from an allogeneic fascial tissue. According toone embodiment, the fascial tissue matrix is derived from a xenogeneicfascial tissue.

According to one embodiment, the at least one fascial tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one fascial tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a fascial tissue-derived growthconductive matrix from which unwanted cells have been removed.

Ligament Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a ligament tissue matrix. According tosome embodiments, the ligament tissue matrix is derived from a ligamenttissue selected from the group consisting of a capsular ligament, anextra-capsular ligament, an intracapsular ligament, a cruciate ligamentand, a combination thereof. The term “ligament” as used herein refers toa band or sheet of fibrous tissue connecting two or more bones,cartilages, or other structures, or serving as support for fasciae ormuscles and a fold of peritoneum supporting any of the abdominalviscera.

According to some embodiments, the ligament tissue matrix is derivedfrom a capsular ligament. According to some embodiments, the ligamenttissue matrix is derived from an extra-capsular ligament. According tosome embodiments, the ligament tissue matrix is derived from anintracapsular ligament. According to some embodiments, the ligamenttissue matrix is derived from a cruciate ligament.

According to some embodiments, the ligament tissue matrix is derivedfrom a ligament-rich body part or at least one fragment thereof.According to some embodiments, the ligament-rich body part is selectedfrom the group consisting of an arm, an elbow, a foot, a hand, a head, aknee, a leg, a neck, a pelvis, a phalange, a thorax, a toe, a wrist and,a combination thereof. According to some embodiments, the ligament-richbody part comprises an arm. According to some embodiments, theligament-rich body part comprises an elbow. According to someembodiments, the ligament-rich body part comprises a foot. According tosome embodiments, the ligament-rich body part comprises a hand.According to some embodiments, the ligament-rich body part comprises ahead. According to some embodiments, the ligament-rich body partcomprises a knee. According to some embodiments, the ligament-rich bodypart comprises a leg. According to some embodiments, the ligament-richbody part comprises a neck. According to some embodiments, theligament-rich body part comprises a pelvis. According to someembodiments, the ligament-rich body part comprises a phalange. Accordingto some embodiments, the ligament-rich body part comprises a thorax.According to some embodiments, the ligament-rich body part comprises atoe. According to some embodiments, the ligament-rich body partcomprises a wrist.

According to some embodiments, the ligament tissue matrix is derivedfrom a ligament organ or at least one fragment thereof. According tosome embodiments, the ligament organ is selected from the groupconsisting of a joint, a mouth, a patella and, a combination thereof.According to some embodiments, the ligament organ comprises a joint.According to some embodiments, the ligament organ comprises a mouth.According to some embodiments, the ligament organ comprises a patella.

According to some embodiments, the ligament tissue matrix is derivedfrom a ligament tissue from a human donor. According to someembodiments, the human donor is a cadaveric donor. According to someembodiments, the human donor is a living donor.

According to one embodiment, the ligament tissue matrix is derived froman autologous ligament tissue. According to one embodiment, the ligamenttissue matrix is derived from an allogeneic ligament tissue. Accordingto one embodiment, the ligament tissue matrix is derived from axenogeneic ligament tissue.

According to one embodiment, the at least one ligament tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one ligament tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a ligament tissue-derived growthconductive matrix from which unwanted cells have been removed.

Mammary Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a mammary tissue matrix. According tosome embodiments, the mammary tissue matrix is derived from a mammaryorgan.

According to some embodiments, the mammary tissue matrix is derived froma mammary tissue of a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, thehuman donor is a living donor.

According to one embodiment, the mammary tissue matrix is derived froman autologous mammary tissue. According to one embodiment, the mammarytissue matrix is derived from an allogeneic mammary tissue. According toone embodiment, the mammary tissue matrix is derived from a xenogeneicmammary tissue.

According to one embodiment, the at least one mammary tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one mammary tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a mammary tissue-derived growthconductive matrix from which unwanted cells have been removed.

Muscle Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a muscle tissue matrix. According tosome embodiments, the muscle tissue matrix is derived from a muscletissue selected from the group consisting of a cardiac muscle tissue, askeletal muscle tissue, a smooth muscle tissue and, a combinationthereof. According to some embodiments, the muscle tissue matrix isderived from a cardiac muscle tissue. According to some embodiments, themuscle tissue matrix is derived from a muscle tissue comprising askeletal muscle tissue. According to some embodiments, the muscle tissuematrix is derived from a smooth muscle tissue.

According to some embodiments, the muscle tissue matrix is derived froma muscle tissue-rich organ or at least one fragment thereof.

According to some embodiments, the muscle tissue-rich organ is selectedfrom the group consisting of a gastrointestinal organ, a skeletal organ,a heart and, a combination thereof. According to some embodiments, themuscle tissue-rich organ comprises a gastrointestinal organ. Accordingto some embodiments, the muscle tissue-rich organ comprises a skeletalorgan. According to some embodiments, the muscle tissue-rich organcomprises a heart.

According to some embodiments, the muscle tissue matrix is derived froma muscle tissue from a human donor. According to some embodiments, thehuman donor is a cadaveric donor. According to some embodiments, thehuman donor is a living donor.

According to one embodiment, the muscle tissue matrix is derived from anautologous muscle tissue. According to one embodiment, the muscle tissuematrix is derived from an allogeneic muscle tissue. According to oneembodiment, the muscle tissue matrix is derived from a xenogeneic muscletissue.

According to one embodiment, the at least one muscle tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one muscle tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a muscle tissue-derived growthconductive matrix from which unwanted cells have been removed.

Nerve Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a nerve tissue matrix. According to someembodiments, the nerve tissue matrix is derived from a nerve tissue froma nerve tissue-rich organ or at least one fragment thereof.

According to some embodiments, the nerve tissue-rich organ is selectedfrom the group consisting of a brain, a spinal cord, and, a combinationthereof. According to some embodiments, the nerve tissue-rich organ is abrain. According to some embodiments, the nerve tissue-rich organ is aspinal cord.

According to some embodiments, the nerve tissue matrix is derived from anerve tissue derived from a human donor. According to some embodiments,the human donor is a cadaveric donor.

According to one embodiment, the nerve tissue matrix is derived from anautologous nerve tissue. According to one embodiment, the nerve tissuematrix is derived from an allogeneic nerve tissue. According to oneembodiment, the nerve tissue matrix is derived from from a xenogeneicnerve tissue.

According to one embodiment, the at least one nerve tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one nerve tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a nerve tissue-derived growthconductive matrix from which unwanted cells have been removed.

Placental Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a placental tissue matrix. According tosome embodiments, the placental tissue matrix is derived from aplacental tissue selected from the group consisting of an amnion tissue,a chorion tissue, an umbilical cord tissue, and a combination thereof.According to some embodiments, the placental tissue matrix is derivedfrom an amnion tissue. According to some embodiments, the placentaltissue matrix is derived from a chorion tissue. According to someembodiments, the placental tissue matrix is derived from a combinationof amnion tissue and chorion tissue. According to some embodiments, theplacental tissue matrix is derived from a combination of amnion tissue,chorion tissue and umbilical cord tissue. According to some embodiments,the placental tissue matrix is derived from an umbilical cord tissue.According to some embodiments, the umbilical cord tissue is selectedfrom the group consisting of an umbilical cord membrane, umbilical cordblood, and a combination thereof.

According to some embodiments, the placental tissue matrix is derivedfrom an isolated placental organ. According to some embodiments, theplacental organ is selected from the group consisting of an amnion, achorion, an umbilical cord, a placenta, and a combination thereof.According to some embodiments, the placental organ is an amnion.According to some embodiments, the placental organ is a chorion.According to some embodiments, the placental organ is a placenta.According to some embodiments, the placental organ is an umbilical cord.

According to some embodiments, the placental tissue matrix is derivedfrom an autologous placental tissue. According to some embodiments, theplacental tissue matrix is derived from an allogeneic placental tissue.According to some embodiments, the placental tissue matrix is derivedfrom a xenogeneic placental tissue.

According to one embodiment, the at least one placental tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one placental tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a placental tissue-derived growthconductive matrix from which unwanted cells have been removed. Accordingto one embodiment, the at least one tissue derived growth-conductivematrix comprises an amnion tissue-derived growth conductive matrix fromwhich unwanted cells have been removed. According to one embodiment, theat least one tissue derived growth-conductive matrix comprises a choriontissue-derived growth conductive matrix from which unwanted cells havebeen removed. According to one embodiment, the at least one tissuederived growth-conductive matrix comprises an umbilical cordtissue-derived growth conductive matrix from which unwanted cells havebeen removed.

Skin Tissue Matrix

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises a skin tissue matrix. According to someembodiments, the skin tissue matrix is derived from a skin tissueselected from the group consisting of an epidermal tissue, a dermaltissue, a basement membrane tissue, and a combination thereof. Accordingto some embodiments, the skin tissue matrix is derived from an epidermaltissue. According to some embodiments, the skin tissue matrix is derivedfrom a dermal tissue. According to some embodiments, the skin tissuematrix is derived from a basement membrane tissue.

According to some embodiments, the skin tissue comprises a tissue matrixderived from a human donor. According to some embodiments, the humandonor is a living donor. According to some embodiments, human donor is acadaveric donor.

According to some embodiments, the skin tissue matrix is derived from anautologous skin tissue. According to some embodiments, the skin tissuematrix is derived from from an allogeneic skin tissue. According to someembodiments, the skin tissue matrix is derived from a xenogeneic skintissue.

According to one embodiment, the at least one skin tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one skin tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a skin tissue-derived growthconductive matrix from which unwanted cells have been removed. Accordingto one embodiment, the at least one tissue derived growth-conductivematrix comprises a dermal tissue-derived growth conductive matrix fromwhich unwanted cells have been removed.

Vascular Tissue Matrix

The term “vasculature” as used herein refers to the vascular network ofa part of the body and its arrangement. The vascular tissue refers tothe tissue comprising the vascular network. The vascular networkcomprises blood vessels, i.e. any vessel conveying blood: arteries,arterioles, capillaries, venules, and veins. An artery is a relativelythick-walled, musclular pulsating vessel conveying blood away from theheart. A vein is a blood vessel carrying blood toward the heart. Botharteries and veins comprises three layers: the tunica intima, the tunicamedia and the tunica adventitia. Veins contain valves that prevent bloodbackflow. The tunica intima, a single layer of simple squamousendothelial cells glued by a polysaccharide intercellular matrix,surrounded by a thin layer of subendothelial connective tissueinterlaced with a number of circularly arranged elastic bands called theinternal elastic lamina; a tunica media, comprising circularly arrangedelastic fiber, connective tissue, polysaccharide substances, and a thickelastic band called the external elastic lamina, and the tunicaadventitia, entirely made of connective tissue. Capillaries comprise alayer of endothelium and connective tissue. According to someembodiments, the tissue comprises vascular tissue.

According to one embodiment, the at least one growth-conductive matrixderived from a tissue comprises an vascular tissue matrix.

According to some embodiments, the vascular tissue matrix is derivedfrom a human donor. According to some embodiments, the human donor is acadaveric donor. According to some embodiments, the human donor is aliving donor.

According to one embodiment, the vascular tissue matrix is derived froman autologous vascular tissue. According to one embodiment, the vasculartissue matrix is derived from an allogeneic vascular tissue. Accordingto one embodiment, the vascular tissue matrix is derived from axenogeneic vascular tissue.

According to one embodiment, the at least one vascular tissue derivedgrowth-conductive matrix comprises a tissuegenic cell niche endogenousto the at least one vascular tissue-derived growth-conductive matrix.

According to one embodiment, the at least one tissue derivedgrowth-conductive matrix comprises a vascular tissue-derived growthconductive matrix from which unwanted cells have been removed. Accordingto one embodiment, the at least one tissue derived growth-conductivematrix comprises an endothelial tissue-derived growth conductive matrixfrom which unwanted cells have been removed.

Tissuegenic Cells

According to one embodiment, the at least one viable population oftissuegenic cells of the implant is adherent to and resident in anendogenous milieu of the growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsadherent to and resident in an endogenous milieu of thegrowth-conductive matrix comprises at least one viable population oftissuegenic cells selected from the group consisting of a viable stemcell population and a viable progenitor cell population. According toone embodiment, the at least one viable population of tissuegenic cellsadherent to and resident in the endogenous milieu of thegrowth-conductive matrix comprises at least one viable stem cellpopulation. According to one embodiment, the at least one viablepopulation of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix comprises at least oneviable progenitor cell population.

According to one embodiment, the at least one viable stem cellpopulation is selected from the group consisting of a viable embryonicstem cell population, a viable pluripotent stem cell population and aviable somatic stem cell population. According to one embodiment, the atleast one viable stem cell population comprises at least one viableembryonic stem cell population. According to one embodiment, the atleast one viable stem cell population comprises at least one viablepluripotent stem cell population. According to one embodiment, the atleast one viable stem cell population comprises at least one viablesomatic stem cell population. According to one embodiment, the at leastone viable pluripotent stem cell population comprises at least oneviable induced pluripotent stem cell (iPSC) population. According to oneembodiment, the at least one viable tissuegenic cell population can bereprogrammed to form at least one viable induced pluripotent stem cell(iPSC) population.

According to one embodiment, the at least one viable somatic stem cellpopulation is selected from the group consisting of a viablehematopoetic stem cell population, a viable mesenchymal stem cellpopulation, a viable neural stem cell population, a viable epithelialstem cell population, a viable lung stem cell, a viable skin stem cellpopulation, and a combination thereof. According to one embodiment, theat least one viable somatic stem cell population comprises at least oneviable hematopoetic stem cell population. According to one embodiment,the at least one viable somatic stem cell population comprises at leastone viable mesenchymal stem cell population. According to oneembodiment, the at least one viable somatic stem cell populationcomprises at least one viable neural stem cell population. According toone embodiment, the at least one viable somatic stem cell populationcomprises at least one viable epithelial stem cell population. Accordingto one embodiment, the at least one viable somatic stem cell populationcomprises at least one viable lung stem cell population. According toone embodiment, the at least one viable somatic stem cell populationcomprises at least one viable skin stem cell population.

According to one embodiment, the at least one viable pluripotent stemcell population is selected from the group consisting of a viablepluripotent stem cell population derived from an adipose tissue, aviable pluripotent stem cell population derived from an amnion tissue, aviable pluripotent stem cell population derived from an artery tissue, aviable pluripotent stem cell population derived from a bone tissue, aviable pluripotent stem cell population derived from a cartilage tissue,a viable pluripotent stem cell population derived from a chorion tissue,a viable pluripotent stem cell population derived from a colon tissue, aviable pluripotent stem cell population derived from a dental tissue, aviable pluripotent stem cell population derived from a dermal tissue, aviable pluripotent stem cell population derived from a duodenal tissue,a viable pluripotent stem cell population derived from an epithelialtissue, a viable pluripotent stem cell population derived from a fascialtissue, a viable pluripotent stem cell population derived from agastrointestinal tissue, a viable pluripotent stem cell populationderived from a growth plate tissue, a viable pluripotent stem cellpopulation derived from an intervertebral disc tissue, a viablepluripotent stem cell population derived from an intestinal mucosal disctissue, a viable pluripotent stem cell population derived from anintestinal serosal tissue, a viable pluripotent stem cell populationderived from a kidney tissue, a viable pluripotent stem cell populationderived from a ligament tissue, a viable pluripotent stem cellpopulation derived from a liver tissue, a viable pluripotent stem cellpopulation derived from a lung tissue, a viable pluripotent stem cellpopulation derived from a mammary tissue, a viable pluripotent stem cellpopulation derived from a meniscal tissue, a viable pluripotent stemcell population derived from a muscle tissue, a viable pluripotent stemcell population derived from a nerve tissue, a viable pluripotent stemcell population derived from an ovarian tissue, a viable pluripotentstem cell population derived from a pancreatic tissue, a viablepluripotent stem cell population derived from a parenchymal organtissue, a viable pluripotent stem cell population derived from apericardial tissue, a viable pluripotent stem cell population derivedfrom a periosteal tissue, a viable pluripotent stem cell populationderived from a peritoneal tissue, a viable pluripotent stem cellpopulation derived from a placental tissue, a viable pluripotent stemcell population derived from a reproductive epithelial tissue, a viablepluripotent stem cell population derived from a respiratory epithelialtissue, a viable pluripotent stem cell population derived from a skintissue, a viable pluripotent stem cell population derived from a spleentissue, a viable pluripotent stem cell population derived from a stomachtissue, a viable pluripotent stem cell population derived from asynovial tissue, a viable pluripotent stem cell population derived froma tendon tissue, a viable pluripotent stem cell population derived froma testes tissue, a viable pluripotent stem cell population derived froman umbilical cord tissue, a viable pluripotent stem cell populationderived from a urological tissue, a viable pluripotent stem cellpopulation derived from a vascular tissue, a viable pluripotent stemcell population derived from a vein tissue, and a combination thereof.According to one embodiment, the at least one viable pluripotent stemcell population comprises a viable pluripotent stem cell populationderived from an adipose tissue. According to one embodiment, the atleast one viable pluripotent stem cell population comprises a viablepluripotent stem cell population derived from an amnion tissue.According to one embodiment, the at least one viable pluripotent stemcell population comprises a viable pluripotent stem cell populationderived from an artery tissue. According to one embodiment, the at leastone viable pluripotent stem cell population comprises a viablepluripotent stem cell population derived from a bone tissue. Accordingto one embodiment, the at least one viable pluripotent stem cellpopulation comprises a viable pluripotent stem cell population derivedfrom a cartilage tissue. According to one embodiment, the at least oneviable pluripotent stem cell population comprises a viable pluripotentstem cell population derived from a chorion tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from a colontissue. According to one embodiment, the at least one viable pluripotentstem cell population comprises a viable pluripotent stem cell populationderived from a dental tissue. According to one embodiment, the at leastone viable pluripotent stem cell population comprises a viablepluripotent stem cell population derived from a dermal tissue. Accordingto one embodiment, the at least one viable pluripotent stem cellpopulation comprises a viable pluripotent stem cell population derivedfrom a duodenal tissue. According to one embodiment, the at least oneviable pluripotent stem cell population comprises a viable pluripotentstem cell population derived from an epithelial tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from afascial tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a gastrointestinal tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from agrowth plate tissue. According to one embodiment, the at least oneviable pluripotent stem cell population comprises a viable pluripotentstem cell population derived from an intervertebral disc tissue.According to one embodiment, the at least one viable pluripotent stemcell population comprises a viable pluripotent stem cell populationderived from an intestinal mucosal tissue. According to one embodiment,the at least one viable pluripotent stem cell population comprises aviable pluripotent stem cell population derived from an intestinalserosal tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a kidney tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from aligament tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a liver tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from a lungtissue. According to one embodiment, the at least one viable pluripotentstem cell population comprises a viable pluripotent stem cell populationderived from a mammary tissue. According to one embodiment, the at leastone viable pluripotent stem cell population comprises a viablepluripotent stem cell population derived from a meniscal tissue.According to one embodiment, the at least one viable pluripotent stemcell population comprises a viable pluripotent stem cell populationderived from a muscle tissue. According to one embodiment, the at leastone viable pluripotent stem cell population comprises a viablepluripotent stem cell population derived from a nerve tissue. Accordingto one embodiment, the at least one viable pluripotent stem cellpopulation comprises a viable pluripotent stem cell population derivedfrom an ovarian tissue. According to one embodiment, the at least oneviable pluripotent stem cell population comprises a viable pluripotentstem cell population derived from a pancreatic tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from aparenchymal organ tissue. According to one embodiment, the at least oneviable pluripotent stem cell population comprises a viable pluripotentstem cell population derived from a pericardial tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from aperiosteal tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a peritoneal tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from aplacental tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a reproductive epithelial tissue. Accordingto one embodiment, the at least one viable pluripotent stem cellpopulation comprises a viable pluripotent stem cell population derivedfrom a respiratory epithelial tissue. According to one embodiment, theat least one viable pluripotent stem cell population comprises a viablepluripotent stem cell population derived from a skin tissue. Accordingto one embodiment, the at least one viable pluripotent stem cellpopulation comprises a viable pluripotent stem cell population derivedfrom a spleen tissue. According to one embodiment, the at least oneviable pluripotent stem cell population comprises a viable pluripotentstem cell population derived from a stomach tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from asynovial tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a tendon tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from atestes tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from an umbilical cord tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from aurological tissue. According to one embodiment, the at least one viablepluripotent stem cell population comprises a viable pluripotent stemcell population derived from a vascular tissue. According to oneembodiment, the at least one viable pluripotent stem cell populationcomprises a viable pluripotent stem cell population derived from a veintissue. According to one embodiment, the at least one viable somaticstem cell population is selected from the group consisting of a viablesomatic stem cell population derived from an adipose tissue, a viablesomatic stem cell population derived from an amnion tissue, a viablesomatic stem cell population derived from an artery tissue, a viablesomatic stem cell population derived from a bone tissue, a viablesomatic stem cell population derived from a cartilage tissue, a viablesomatic stem cell population derived from a chorion tissue, a viablesomatic stem cell population derived from a colon tissue, a viablesomatic stem cell population derived from a dental tissue, a viablesomatic stem cell population derived from a dermal tissue, a viablesomatic stem cell population derived from a duodenal tissue, a viablesomatic stem cell population derived from an epithelial tissue, a viablesomatic stem cell population derived from a fascial tissue, a viablesomatic stem cell population derived from a gastrointestinal tissue, aviable somatic stem cell population derived from a growth plate tissue,a viable somatic stem cell population derived from an intervertebraldisc tissue, a viable somatic stem cell population derived from anintestinal mucosal disc tissue, a viable somatic stem cell populationderived from an intestinal serosal tissue, a viable somatic stem cellpopulation derived from a kidney tissue, a viable somatic stem cellpopulation derived from a ligament tissue, a viable somatic stem cellpopulation derived from a liver tissue, a viable somatic stem cellpopulation derived from a lung tissue, a viable somatic stem cellpopulation derived from a mammary tissue, a viable somatic stem cellpopulation derived from a meniscal tissue, a viable somatic stem cellpopulation derived from a muscle tissue, a viable somatic stem cellpopulation derived from a nerve tissue, a viable somatic stem cellpopulation derived from an ovarian tissue, a viable somatic stem cellpopulation derived from a pancreatic tissue, a viable somatic stem cellpopulation derived from a parenchymal organ tissue, a viable somaticstem cell population derived from a pericardial tissue, a viable somaticstem cell population derived from a periosteal tissue, a viable somaticstem cell population derived from a peritoneal tissue, a viable somaticstem cell population derived from a placental tissue, a viable somaticstem cell population derived from a reproductive epithelial tissue, aviable somatic stem cell population derived from a respiratoryepithelial tissue, a viable somatic stem cell population derived from askin tissue, a viable somatic stem cell population derived from a spleentissue, a viable somatic stem cell population derived from a stomachtissue, a viable somatic stem cell population derived from a synovialtissue, a viable somatic stem cell population derived from a tendontissue, a viable somatic stem cell population derived from a testestissue, a viable somatic stem cell population derived from an umbilicalcord tissue, a viable somatic stem cell population derived from aurological tissue, a viable somatic stem cell population derived from avascular tissue, a viable somatic stem cell population derived from avein tissue, and a combination thereof. According to one embodiment, theat least one viable somatic stem cell population comprises a viablesomatic stem cell population derived from an adipose tissue. Accordingto one embodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from an amniontissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from an artery tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from a bone tissue. According to one embodiment,the at least one viable somatic stem cell population comprises a viablesomatic stem cell population derived from a cartilage tissue. Accordingto one embodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a choriontissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a colon tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from a dental tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a dermaltissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a duodenal tissue. According to one embodiment, the atleast one viable somatic stem cell population comprises a viable somaticstem cell population derived from an epithelial tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a fascialtissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a gastrointestinal tissue. According to one embodiment, theat least one viable somatic stem cell population comprises a viablesomatic stem cell population derived from a growth plate tissue.According to one embodiment, the at least one viable somatic stem cellpopulation comprises a viable somatic stem cell population derived froman intervertebral disc tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from an intestinal mucosal tissue. According toone embodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from anintestinal serosal tissue. According to one embodiment, the at least oneviable somatic stem cell population comprises a viable somatic stem cellpopulation derived from a kidney tissue. According to one embodiment,the at least one viable somatic stem cell population comprises a viablesomatic stem cell population derived from a ligament tissue. Accordingto one embodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a livertissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a lung tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from a mammary tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a meniscaltissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a muscle tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from a nerve tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from an ovariantissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a pancreatic tissue. According to one embodiment, the atleast one viable somatic stem cell population comprises a viable somaticstem cell population derived from a parenchymal organ tissue. Accordingto one embodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from apericardial tissue. According to one embodiment, the at least one viablesomatic stem cell population comprises a viable somatic stem cellpopulation derived from a periosteal tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from aperitoneal tissue. According to one embodiment, the at least one viablesomatic stem cell population comprises a viable somatic stem cellpopulation derived from a placental tissue. According to one embodiment,the at least one viable somatic stem cell population comprises a viablesomatic stem cell population derived from a reproductive epithelialtissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a respiratory epithelial tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a skintissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a spleen tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from a stomach tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a synovialtissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a tendon tissue. According to one embodiment, the at leastone viable somatic stem cell population comprises a viable somatic stemcell population derived from a testes tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from anumbilical cord tissue. According to one embodiment, the at least oneviable somatic stem cell population comprises a viable somatic stem cellpopulation derived from a urological tissue. According to oneembodiment, the at least one viable somatic stem cell populationcomprises a viable somatic stem cell population derived from a vasculartissue. According to one embodiment, the at least one viable somaticstem cell population comprises a viable somatic stem cell populationderived from a vein tissue.

According to one embodiment, the at least one viable progenitor stemcell population is selected from the group consisting of a viableprogenitor stem cell population derived from an adipose tissue, a viableprogenitor stem cell population derived from an amnion tissue, a viableprogenitor stem cell population derived from an artery tissue, a viableprogenitor stem cell population derived from a bone tissue, a viableprogenitor stem cell population derived from a cartilage tissue, aviable progenitor stem cell population derived from a chorion tissue, aviable progenitor stem cell population derived from a colon tissue, aviable progenitor stem cell population derived from a dental tissue, aviable progenitor stem cell population derived from a dermal tissue, aviable progenitor stem cell population derived from a duodenal tissue, aviable progenitor stem cell population derived from an epithelialtissue, a viable progenitor stem cell population derived from a fascialtissue, a viable progenitor stem cell population derived from agastrointestinal tissue, a viable progenitor stem cell populationderived from a growth plate tissue, a viable progenitor stem cellpopulation derived from an intervertebral disc tissue, a viableprogenitor stem cell population derived from an intestinal mucosal disctissue, a viable progenitor stem cell population derived from anintestinal serosal tissue, a viable progenitor stem cell populationderived from a kidney tissue, a viable progenitor stem cell populationderived from a ligament tissue, a viable progenitor stem cell populationderived from a liver tissue, a viable progenitor stem cell populationderived from a lung tissue, a viable progenitor stem cell populationderived from a mammary tissue, a viable progenitor stem cell populationderived from a meniscal tissue, a viable progenitor stem cell populationderived from a muscle tissue, a viable progenitor stem cell populationderived from a nerve tissue, a viable progenitor stem cell populationderived from an ovarian tissue, a viable progenitor stem cell populationderived from a pancreatic tissue, a viable progenitor stem cellpopulation derived from a parenchymal organ tissue, a viable progenitorstem cell population derived from a pericardial tissue, a viableprogenitor stem cell population derived from a periosteal tissue, aviable progenitor stem cell population derived from a peritoneal tissue,a viable progenitor stem cell population derived from a placentaltissue, a viable progenitor stem cell population derived from areproductive epithelial tissue, a viable progenitor stem cell populationderived from a respiratory epithelial tissue, a viable progenitor stemcell population derived from a skin tissue, a viable progenitor stemcell population derived from a spleen tissue, a viable progenitor stemcell population derived from a stomach tissue, a viable progenitor stemcell population derived from a synovial tissue, a viable progenitor stemcell population derived from a tendon tissue, a viable progenitor stemcell population derived from a testes tissue, a viable progenitor stemcell population derived from an umbilical cord tissue, a viableprogenitor stem cell population derived from a urological tissue, aviable progenitor stem cell population derived from a vascular tissue, aviable progenitor stem cell population derived from a vein tissue, and acombination thereof.

According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from an adipose tissue. According to one embodiment, the atleast one viable progenitor stem cell population comprises a viableprogenitor stem cell population derived from an amnion tissue. Accordingto one embodiment, the at least one viable progenitor stem cellpopulation comprises a viable progenitor stem cell population derivedfrom an artery tissue. According to one embodiment, the at least oneviable progenitor stem cell population comprises a viable progenitorstem cell population derived from a bone tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from acartilage tissue. According to one embodiment, the at least one viableprogenitor stem cell population comprises a viable progenitor stem cellpopulation derived from a chorion tissue. According to one embodiment,the at least one viable progenitor stem cell population comprises aviable progenitor stem cell population derived from a colon tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from a dental tissue. According to one embodiment, the at leastone viable progenitor stem cell population comprises a viable progenitorstem cell population derived from a dermal tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from aduodenal tissue. According to one embodiment, the at least one viableprogenitor stem cell population comprises a viable progenitor stem cellpopulation derived from an epithelial tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from afascial tissue. According to one embodiment, the at least one viableprogenitor stem cell population comprises a viable progenitor stem cellpopulation derived from a gastrointestinal tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from a growthplate tissue. According to one embodiment, the at least one viableprogenitor stem cell population comprises a viable progenitor stem cellpopulation derived from an intervertebral disc tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from anintestinal mucosal tissue. According to one embodiment, the at least oneviable progenitor stem cell population comprises a viable progenitorstem cell population derived from an intestinal serosal tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from a kidney tissue. According to one embodiment, the at leastone viable progenitor stem cell population comprises a viable progenitorstem cell population derived from a ligament tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from a livertissue. According to one embodiment, the at least one viable progenitorstem cell population comprises a viable progenitor stem cell populationderived from a lung tissue. According to one embodiment, the at leastone viable progenitor stem cell population comprises a viable progenitorstem cell population derived from a mammary tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from ameniscal tissue. According to one embodiment, the at least one viableprogenitor stem cell population comprises a viable progenitor stem cellpopulation derived from a muscle tissue. According to one embodiment,the at least one viable progenitor stem cell population comprises aviable progenitor stem cell population derived from a nerve tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from an ovarian tissue. According to one embodiment, the atleast one viable progenitor stem cell population comprises a viableprogenitor stem cell population derived from a pancreatic tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from a parenchymal organ tissue. According to one embodiment,the at least one viable progenitor stem cell population comprises aviable progenitor stem cell population derived from a pericardialtissue. According to one embodiment, the at least one viable progenitorstem cell population comprises a viable progenitor stem cell populationderived from a periosteal tissue. According to one embodiment, the atleast one viable progenitor stem cell population comprises a viableprogenitor stem cell population derived from a peritoneal tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from a placental tissue. According to one embodiment, the atleast one viable progenitor stem cell population comprises a viableprogenitor stem cell population derived from a reproductive epithelialtissue. According to one embodiment, the at least one viable progenitorstem cell population comprises a viable progenitor stem cell populationderived from a respiratory epithelial tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from a skintissue. According to one embodiment, the at least one viable progenitorstem cell population comprises a viable progenitor stem cell populationderived from a spleen tissue. According to one embodiment, the at leastone viable progenitor stem cell population comprises a viable progenitorstem cell population derived from a stomach tissue. According to oneembodiment, the at least one viable progenitor stem cell populationcomprises a viable progenitor stem cell population derived from asynovial tissue. According to one embodiment, the at least one viableprogenitor stem cell population comprises a viable progenitor stem cellpopulation derived from a tendon tissue. According to one embodiment,the at least one viable progenitor stem cell population comprises aviable progenitor stem cell population derived from a testes tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from an umbilical cord tissue. According to one embodiment, theat least one viable progenitor stem cell population comprises a viableprogenitor stem cell population derived from a urological tissue.According to one embodiment, the at least one viable progenitor stemcell population comprises a viable progenitor stem cell populationderived from a vascular tissue. According to one embodiment, the atleast one viable progenitor stem cell population comprises a viableprogenitor stem cell population derived from a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells is capable of migrating from or to the at least onegrowth-conductive matrix.

According to some embodiments, the at least one viable population oftissuegenic cells adherent to and resident in the endogenous milieu ofthe growth conductive matrix is immune privileged. The term “immuneprivileged” as used herein refers to a characteristic of tissuegeniccells by which there is no induction of an immune response upontransplantation of such cells of such cells into an allogeneic host.

Growth-Inductive Factors

Evidence supports the notion that stem cells can adjust their propertiesaccording to their surroundings and select specific lineages accordingto cues they receive from their niche. It follows that in order for atissuegenic cell therapy to be successful in the repair of a specifictissue type, the microenvironment of the tissuegenic cells should bedesigned to relay the appropriate chemical and physical signals to them.

According to one embodiment, the implant further comprises at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductive component is tissue-derived. According to oneembodiment, the at least one growth-inductive component comprisesinducible pluripotent stem cells (iPSCs). According to one embodiment,the at least one growth-inductive component originates from a componentof the tissue-derived growth-inductive component other than cells.According to one embodiment, the at least one growth-inductive factor isendogenous to the at least one growth-conductive matrix. According toone embodiment, the tissuegenic cells adherent to and resident in theendogenous milieu of the growth conductive matrix secrete the at leastone growth-inductive component. According to one embodiment, the atleast one growth-inductive component is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells. According to another embodiment, agrowth-inductive matrix (e.g., an osteoinductive matrix) includes ademineralized bone matrix, which may be prepared from one or more of thebone tissue matrix types and sources of bone tissue matrix describedabove with regard to growth-conductive matrices that include bone tissuematrices. According to one embodiment, the at least one growth-inductivecomponent is a growth-inductivefactor. According to some suchembodiments, the at least one growth-inductivefactor is selected fromthe group consisting of a bone morphogenic protein (BMP), a fibroblastgrowth factor (FGF), an insulin-like growth factor (IGF), aplatelet-derived growth factor (PDGF), a transforming growth factor-β(TGF-β), a neural epidermal growth-factor-like 1 (NELL-1), and acombination thereof. According to one embodiment, the at least oneviable population of tissuegenic cells secretes the at least onegrowth-inductivefactor. According to some such embodiments, the at leastone growth-inductivefactor comprises a bone morphogenic protein.According to some such embodiments, the at least onegrowth-inductivefactor comprises a fibroblast growth factor. Accordingto some such embodiments, the at least one growth-inductivefactorcomprises an insulin-like growth factor. According to some suchembodiments, the at least one growth-inductive factor comprises aplatelet-derived growth factor. According to some such embodiments, theat least one growth-inductivefactor comprises a transforming growthfactor-13. According to some such embodiments, the at least onegrowth-inductivefactor comprises a neural epidermal growth-factor-like1.

Target Lineage of Tissuegenic Cells

According to one embodiment, the at least one viable population oftissuegenic cells is capable of differentiating into cells of at leastone embryonic lineage. According to one embodiment, the embryoniclineage is selected from the group consisting of an ectodermal lineage,a mesodermal lineage and an endodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into cells of an ectodermal lineage.According to one embodiment, the at least one viable population oftissuegenic cells is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells is capable of differentiating into cellsof an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a target tissue. Accordingto one embodiment, the target tissue is selected from the groupconsisting of an adipose tissue, an amnion tissue, an artery tissue, abone tissue, a cartilage tissue, a chorion tissue, a colon tissue, adental tissue, a dermal tissue, a duodenal tissue, an endothelialtissue, an epithelial tissue, a fascial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating an adipose tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating an amnion tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells is capable of regenerating an artery tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a bone tissue. According to one embodiment, theat least one viable population of tissuegenic cells is capable ofregenerating a cartilage tissue. According to one embodiment, the atleast one viable population of tissuegenic cells is capable ofregenerating a chorion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells is capable of regenerating acolon tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells is capable of regenerating a dentaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of regenerating a dermal tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a duodenal tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating an endothelial tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a fascial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells is capable of regenerating a gastrointestinal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of regenerating a growth plate tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating an intestinal mucosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a kidney tissue. According to one embodiment,the at least one viable population of tissuegenic cells is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells is capable ofregenerating a liver tissue. According to one embodiment, the at leastone viable population of tissuegenic cells is capable of regenerating alung tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells is capable of regenerating a meniscaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of regenerating a muscle tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a nerve tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells is capable of regenerating an ovarian tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a parenchymal organ tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a pericardial tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a periosteal tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of regenerating a skin tissue. According to one embodiment, theat least one viable population of tissuegenic cells is capable ofregenerating a spleen tissue. According to one embodiment, the at leastone viable population of tissuegenic cells is capable of regenerating asynovial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells is capable of regenerating a tendontissue. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a urological tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a vascular tissue.According to one embodiment, the at least one viable population oftissuegenic cells is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells is capable of differentiating into a target tissuecell lineage. According to one embodiment, the target tissue celllineage is selected from the group consisting of an adipose celllineage, an amnion cell lineage, an artery cell lineage, a bone celllineage, a cartilage cell lineage, a dental cell lineage, a dermal celllineage, a duodenal cell lineage, an endothelial lineage, an epithelialcell lineage, a fascial cell lineage, a gastrointestinal cell lineage, agrowth plate cell lineage, an intervertebral disc cell lineage, anintestinal mucosal cell lineage, an intestinal serosal cell lineage, akidney cell lineage, a ligament cell lineage, a liver cell lineage, alung cell lineage, a meniscal cell lineage, a muscle cell lineage, anerve cell lineage, an ovarian cell lineage, a pancreatic cell lineage,a parenchymal organ cell lineage, a pericardial cell lineage, aperiosteal cell lineage, a peritoneal cell lineage, a skin cell lineage,a spleen cell lineage, a synovial cell lineage, a tendon cell lineage, atestes cell lineage, a urological cell lineage, a vascular cell lineage,a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells is capable of differentiating into an adipose celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into an amnion celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into an artery celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a bone celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a cartilage celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a chorion celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a colon celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a dental celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a dermal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a duodenal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into an endothelialcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a fascial cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells is capable of differentiatinginto a gastrointestinal cell lineage. According to one embodiment, theat least one viable population of tissuegenic cells is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into an intervertebral disc cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells is capable of differentiating into an intestinalmucosal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto an intestinal serosal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells is capable ofdifferentiating into a kidney cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a liver cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a meniscal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of differentiating into a muscle cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of differentiating into a nerve cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of differentiating into an ovarian cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of differentiating into a pancreatic cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells is capable of differentiating into a parenchymal organ celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells is capable of differentiating into a pericardialcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells is capable ofdifferentiating into a skin cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells is capable ofdifferentiating into a spleen cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells is capable ofdifferentiating into a synovial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a tendon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a urological cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsis capable of differentiating into a vein cell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells comprises a viable expanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of migrating from orto the at least one growth-conductive matrix. According to someembodiments, the at least one viable population of tissuegenic cellsadherent to and resident in the endogenous milieu of the growthconductive matrix is immune privileged. The term “immune privileged” asused herein refers to the characteristic of tissuegenic cells by whichthere is no induction of an immune response upon transplantation of suchcells.

According to one embodiment, the bone cell lineage is selected from thegroup consisting of an osteoprogenitor cell lineage, an osteoblastlineage, an osteocyte lineage, and an osteoclast lineage. According toone embodiment, the bone cell lineage comprises an osteoprogenitor celllineage. According to one embodiment, the bone cell lineage comprises anosteoblast lineage. According to one embodiment, the bone cell lineagecomprises an osteocyte lineage.

According to one embodiment, the cartilage cell lineage is selected fromthe group consisting of a chondrocyte lineage, a chondroblast lineageand a chondroclast lineage. According to one embodiment, the cartilagecell lineage comprises a chondrocyte lineage. According to oneembodiment, the cartilage cell lineage comprises a chondroblast lineage.According to one embodiment, the cartilage cell lineage comprises achondroclast lineage.

According to one embodiment, the dental cell lineage is selected fromthe group consisting of an ameloblast lineage, an odontoblast lineage, acementoblast and a nerve cell lineage. According to one embodiment, thedental cell lineage comprises an ameloblast lineage. According to oneembodiment, the dental cell lineage comprises an odontoblast lineage.According to one embodiment, the dental cell lineage comprises acementoblast lineage. According to one embodiment, the dental celllineage comprises a nerve cell lineage.

According to one embodiment, the intervertebral disc cell lineagecomprises a cartilage cell lineage. According to one embodiment, theintervertebral disc cell lineage is selected from the group consistingof an annulus fibrosus lineage, a nucleus pulposus lineage and anendplate lineage. According to one embodiment, the intervertebral disccell lineage comprises an annulus fibrosus cell lineage. According toone embodiment, the intervertebral disc cell lineage comprises a nucleuspulposus cell lineage. According to one embodiment, the intervertebraldisc cell embodiment comprises an endplate lineage.

According to one embodiment, the gastrointestinal cell lineage comprisesa gastrointestinal epithelial cell lineage. According to one embodiment,the gastrointestinal epithelial cell lineage is selected from the groupconsisting of a columnar epithelial cell lineage, a goblet cell lineage,an enteroendocrine chromaffin cell lineage and a Paneth cell lineage.According to one embodiment, the gastrointestinal epithelial celllineage comprises a columnar epithelial cell lineage. According to oneembodiment, the gastrointestinal epithelial cell lineage comprises agoblet cell lineage. According to one embodiment, the gastrointestinalepithelial cell lineage comprises an enteroendocrine chromaffin celllineage. According to one embodiment, the gastrointestinal epithelialcell lineage comprises a Paneth cell lineage.

According to one embodiment, the growth plate cell lineage comprises acartilage cell lineage.

According to one embodiment, the ligament cell lineage comprises afibroblast lineage. According to one embodiment, the connective tissuecell lineage is selected from the group consisting of a fibroblastlineage, a macrophage lineage, and a mast cell lineage. According to oneembodiment, the connective tissue cell lineage comprises a fibroblastlineage. According to one embodiment, the connective tissue cell lineagecomprises a macrophage lineage. According to one embodiment, theconnective tissue cell lineage comprises a mast cell lineage.

According to one embodiment, the liver cell lineage comprises ahepatocyte lineage.

According to one embodiment, the meniscal cell lineage comprises acartilage cell lineage.

According to one embodiment, the lung cell lineage comprises anepithelial cell lineage. According to one embodiment, the lung celllineage comprises a vascular cell lineage.

According to one embodiment, the muscle cell lineage is selected fromthe group consisting of a smooth muscle cell lineage, a skeletal musclecell lineage and a cardiomyocyte lineage. According to one embodiment,the muscle cell lineage comprises a smooth muscle cell lineage.According to one embodiment, the muscle cell lineage comprises askeletal muscle cell lineage. According to one embodiment, the musclecell lineage comprises a cardiomyocyte lineage.

According to one embodiment, the nerve cell lineage is selected from thegroup consisting of an astrocyte lineage, a dendritic cell lineage, aneuroglial cell lineage, and a neuron lineage. According to oneembodiment, the nerve cell lineage comprises an astrocyte lineage.According to one embodiment, the nerve cell lineage comprises adendritic cell lineage. According to one embodiment, the nerve celllineage comprises a neuroglial cell lineage. According to oneembodiment, the nerve cell lineage comprises a neuron lineage.

According to one embodiment, the periosteal cell lineage comprises aconnective tissue cell lineage.

According to one embodiment, the skin cell lineage is selected from agroup consisting of an epidermal cell lineage and a dermal cell lineage.According to one embodiment, the skin cell lineage comprises anepidermal cell lineage. According to one embodiment, the skin celllineage comprises a dermal cell lineage. According to one embodiment,the skin cell lineage is selected from a group consisting of afibroblast lineage, a keratinocyte lineage, a macrophage lineage and amast cell lineage. According to one embodiment, the skin cell lineagecomprises a fibroblast lineage. According to one embodiment, the skincell lineage comprises a keratinocyte lineage. According to oneembodiment, the skin cell lineage comprises a macrophage lineage.According to one embodiment, the skin cell lineage comprises a mast celllineage.

According to one embodiment, the synovial cell lineage comprises aconnective tissue cell lineage. According to one embodiment, theconnective tissue cell lineage is selected from the group consisting ofa fibroblast lineage, a macrophage lineage, and a mast cell lineage.According to one embodiment, the connective tissue cell lineagecomprises a fibroblast lineage. According to one embodiment, theconnective tissue cell lineage comprises a macrophage lineage. Accordingto one embodiment, the connective tissue cell lineage comprises a mastcell lineage.

According to one embodiment, the tendon cell lineage comprises aconnective tissue cell lineage. According to one embodiment, theconnective tissue cell lineage is selected from the group consisting ofa fibroblast lineage, a macrophage lineage, and a mast cell lineage.According to one embodiment, the connective tissue cell lineagecomprises a fibroblast lineage. According to one embodiment, theconnective tissue cell lineage comprises a macrophage lineage. Accordingto one embodiment, the connective tissue cell lineage comprises a mastcell lineage. According to one embodiment, the tendon cell lineagecomprises a tenocyte lineage. According to one embodiment, theconnective tissue cell lineage comprises a mast cell lineage.

According to one embodiment, the vascular cell lineage comprises aconnective tissue cell lineage. According to one embodiment, theconnective tissue cell lineage is selected from the group consisting ofa fibroblast lineage, a macrophage lineage, and a mast cell lineage.According to one embodiment, the connective tissue cell lineagecomprises a fibroblast lineage. According to one embodiment, theconnective tissue cell lineage comprises a macrophage lineage. Accordingto one embodiment, the connective tissue cell lineage comprises a mastcell lineage. According to one embodiment, the tendon cell lineagecomprises an endothelial lineage.

Tissuegenic Cells Derived from Adipose Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is selected from the groupconsisting of a viable population of pluripotent stem cells, a viablepopulation of mesenchymal stem cells, a viable population ofadipose-derived stem cells, and a viable population of adipose-derivedprogenitor cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is a viablepluripotent stem cell population. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is a viable mesenchymal stem cell population. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is a viable adipose-derived stem cellpopulation. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is a viableadipose-derived progenitor cell population.

According to one embodiment, the at least one viable population oftissuegenic cells from adipose tissue secretes at least onegrowth-inductive factor. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissuesecretes at least one growth-inductive factor such as an adipokineAccording to one embodiment, the adipokine is selected from the groupconsisting of HGF, VEGF, Flt-3 ligand, G-CSF, GM-CSF, IL-7, IL-12,M-CSF, SCF, IL-1alpha, IL-6, IL-8, IL-11, LIF, and TNF-alpha. Otherexemplary adipokines are listed in Kilroy et. al. (2007), J. Cell.Physiol. 212: 702-709, the entire contents of which are incorporated byreference herein. According to one embodiment, the adipokine is anadiponectin. According to one embodiment, the adipokine is a leptin.According to one embodiment, the adipokine is an IL-6. According to oneembodiment, the adipokine is an IL-7. According to one embodiment, theadipokine is an IL-8. According to one embodiment, the adipokine is aMCP-1. According to one embodiment, the adipokine is a GRO. According toone embodiment, the adipokine is an angiogenin. According to oneembodiment, the adipokine is a HGF. According to one embodiment, theadipokine is a VEGF. According to one embodiment, the adipokine is aTIMP-1. According to one embodiment, the adipokine is a TIMP-2. Otherexemplary adipokines, as used in this invention, are listed in Halberget al. (2008), Endocrinol. Metab. Clin. North Am., 37(3): 753-767 and inKlimkakova et al. (2007), Biochem. Biophys. Res. Commun., 358: 897-902,which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into cells ofa mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable of regeneratinga target tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof regenerating an amnion tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is capable of regenerating an artery tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating a bone tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable of regeneratinga cartilage tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof regenerating a chorion tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is capable of regenerating a colon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating a dental tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable of regeneratinga dermal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof regenerating a duodenal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is capable of regenerating an endothelial tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating agastrointestinal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of regenerating a growth plate tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is is capable of regenerating anintervertebral disc tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from adipose tissueis is capable of regenerating an intestinal mucosal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is is capable of regenerating an intestinalserosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is iscapable of regenerating a kidney tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromadipose tissue is capable of regenerating a ligament tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from adipose tissue is capable of regenerating a livertissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from adipose tissue is capable ofregenerating a lung tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from adipose tissueis capable of regenerating a meniscal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating a muscle tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable of regeneratinga nerve tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof regenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof regenerating a pericardial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from adiposetissue is capable of regenerating a periosteal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating a peritonealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from adipose tissue is capable ofregenerating a skin tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from adipose tissueis capable of regenerating a spleen tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromadipose tissue is capable of regenerating a synovial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from adipose tissue is capable of regenerating a tendontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from adipose tissue is capable ofregenerating a testes tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from adipose tissueis capable of regenerating a urological tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from adipose tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from adipose tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from adipose tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from adiposetissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from adipose tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from adipose tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue is capable of differentiating into a veincell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue can bereprogrammed to form at least one viable induced pluripotent stem cell(iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromadipose tissue differentiates along a chondrogenic lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from adipose tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from adipose tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from adipose tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells of such cells.

Tissuegenic Cells Derived from Bone Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is selected from the groupconsisting of a viable population of bone-derived mesenchymal stemcells, a viable population of osteoprogenitor cells, or a combinationthereof. According to one embodiment, the at least one viable populationof tissuegenic cells derived from bone tissue is a viable mesenchymalstem cell population. According to one embodiment, the at least oneviable population of tissuegenic cells derived from bone tissue is aviable osteoprogenitor cell population.

According to one embodiment, the at least one viable population oftissuegenic cells from bone tissue secretes at least onegrowth-inductive factor. Examples of growth-inductive factors secretedby bone tissue include, but are not limited to, Bone MorphogenicProteins (BMPs), Epidermal Growth Factors (EGFs), Fibroblast GrowthFactors (FGFs), Platelet-Derived Growth Factors (PDGFs), Insulin-likeGrowth Factor-1 (IGF-1), Transforming Growth Factors (TGFs),Bone-Derived Growth Factors (BDGFs), Cartilage-Derived Growth Factor(CDGF), Skeletal Growth Factor (hSGF), Interleukin-1 (IL-1), andmacrophage-derived factors.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto cells of at least one embryonic lineage. According to oneembodiment, the embryonic lineage is selected from the group consistingof an ectodermal lineage, a mesodermal lineage and an endodermallineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from bone tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating atarget tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating anadipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of regenerating a cartilage tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating achorion tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissue iscapable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of regenerating a dermal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of regenerating a duodenal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating anendothelial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofregenerating a gastrointestinal tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from bonetissue is capable of regenerating a growth plate tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is is capable of regenerating an intervertebraldisc tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is is capableof regenerating an intestinal mucosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is is capable of regenerating an intestinalserosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is is capableof regenerating a kidney tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of regenerating a ligament tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of regenerating a liver tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating alung tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from adipose tissue is capableof regenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating anovarian tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofregenerating a pancreatic tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of regenerating a parenchymal organ tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from bone tissue is capable of regenerating a pericardialtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from bone tissue is capable of regeneratinga periosteal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from bone tissue iscapable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating aspleen tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of regenerating aurological tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofregenerating a vascular tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto a target tissue cell lineage. According to one embodiment, thetarget tissue cell lineage is selected from the group consisting of anadipose cell lineage, an amnion cell lineage, an artery cell lineage, abone cell lineage, a cartilage cell lineage, a dental cell lineage, adermal cell lineage, a duodenal cell lineage, an endothelial lineage, anepithelial cell lineage, a gastrointestinal cell lineage, a growth platecell lineage, an intervertebral disc cell lineage, an intestinal mucosalcell lineage, an intestinal serosal cell lineage, a kidney cell lineage,a ligament cell lineage, a liver cell lineage, a lung cell lineage, ameniscal cell lineage. a muscle cell lineage, a nerve cell lineage, anovarian cell lineage, a pancreatic cell lineage, a parenchymal organcell lineage, a pericardial cell lineage, a periosteal cell lineage, aperitoneal cell lineage, a skin cell lineage, a spleen cell lineage, asynovial cell lineage, a tendon cell lineage, a testes cell lineage, aurological cell lineage, a vascular cell lineage, a vein cell lineage,and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto an adipose cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissue iscapable of differentiating into an amnion cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into an arterycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into a bone cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of differentiating into a cartilage cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto a chorion cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of differentiating into a duodenal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto an endothelial cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of differentiating into an epithelial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto a gastrointestinal cell lineage. According to one embodiment, theat least one viable population of tissuegenic cells derived from bonetissue is capable of differentiating into a growth plate cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto an intervertebral disc cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of differentiating into an intestinal mucosalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into a liver celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from bone tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of differentiating into a meniscal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto a muscle cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissue iscapable of differentiating into a nerve cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into an ovariancell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into a pancreatic cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into aparenchymal organ cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from bonetissue is capable of differentiating into a pericardial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto a periosteal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells is capable ofdifferentiating into a peritoneal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into a skin celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from bone tissue is capable ofdifferentiating into a spleen cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frombone tissue is capable of differentiating into a synovial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of differentiatinginto a tendon cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into a urologicalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue is capable of differentiating into a vein celllineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue differentiatesalong an adipogenic lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from bone tissuedifferentiates along a chondrogenic lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from bone tissue is capable of migrating fromor to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from bone tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from bone tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from bone tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells of such cells.

Tissuegenic Cells Derived from Cartilage Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is selected from thegroup consisting of a viable population of cartilage-derived mesenchymalstem cells and a viable population of cartilage derived progenitorcells. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is a viablecartilage-derived mesenchymal stem cell population. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is a viable cartilage-derived progenitorcell population. Exemplary tissuegenic cells derived from cartilagetissue are listed in Asalameh et al., Arthritis & Rheumatism (2004),50(5): 1522-1532, Peng et al., Stem Cells and Development (2008), 17:761-774, Hiraoka et al., Biorheology (2006), 43: 447-454, Karlsson etal., 2009, J. Anat. 215(3): 355-63 and Grogan et al., Arthritis Res.Ther. (2009), 11(3): R85-R97, the entire contents of which areincorporated herein by reference. Exemplary tissuegenic cells derivedfrom cartilage tissue of intervertebral discs are listed in Henrikssonet al. (2009), SPINE, 34(21): 2278-2287, the entire contents of whichare incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from cartilage tissue secretes at least onegrowth-inductive factor. Exemplary growth-inductive components include,but are not limited to, ions (e.g., calcium); steroids (e.g.,estrogens); terpenoids (e.g., retinoic acid); peptides (e.g.,Parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP),insulin growth factors (e.g., TGFβ hormones, including, withoutlimitation, BMPs, IGF-1, VEGF, PDGF, FGF); transcription factors (e.g.,Wnt, SOX-9); eicosanoids (e.g., prostaglandins); catabolic interleukins(e.g., IL-1); and anabolic interleukins (e.g., IL-6, IL-4 and IL-10).Other growth-inductive components are listed in Gaissmaier et al.(2008), Int. J. Care Injured, 39S1: S88-S96, the entire contents ofwhich are incorporated by reference herein. According to one embodiment,the at least one viable population of tissuegenic cells derived fromcartilage tissue is capable of differentiating into cells of at leastone embryonic lineage. According to one embodiment, the embryoniclineage is selected from the group consisting of an ectodermal lineage,a mesodermal lineage and an endodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into cellsof an ectodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage t tissueis capable of differentiating into cells of a mesodermal lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofdifferentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from cartilagetissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromcartilage tissue is capable of regenerating a dermal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from cartilage tissue is capable of regenerating aduodenal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from cartilage tissue is capableof regenerating an endothelial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived fromcartilage tissue is capable of regenerating a gastrointestinal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofregenerating a growth plate tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from cartilagetissue is is capable of regenerating an intervertebral disc tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is is capable ofregenerating an intestinal mucosal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromcartilage tissue is is capable of regenerating an intestinal serosaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is is capable ofregenerating a kidney tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating a ligament tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a liver tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofregenerating a lung tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating a meniscal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a muscletissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofregenerating a nerve tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating an ovarian tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a pancreatictissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofregenerating a parenchymal organ tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromcartilage tissue is capable of regenerating a pericardial tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofregenerating a periosteal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from cartilagetissue is capable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofregenerating a spleen tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating a synovial tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a tendontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofregenerating a testes tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of regenerating a urological tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from cartilage tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into adental cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from cartilage tissue is capable of differentiating intoan epithelial cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from cartilage tissueis capable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from cartilagetissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from cartilage tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into akidney cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a ligament cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from cartilage tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from cartilage tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from cartilagetissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from cartilage tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into atendon cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from cartilage tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromcartilage tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from cartilage tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from cartilage tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from cartilage tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from cartilage tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from cartilage tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Tissuegenic Cells Derived from Dental Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is selected from the groupconsisting of a viable population of stem cells of apical papilla(SCAP), a viable population of dental pulp stem cells (DPSCs), a viablepopulation of stem cells from exfoliated deciduous teeth (SHED), aviable population of periodontal ligament stem cells (PDLSCs), and aviable population of dental follicle stem cells (DFSCs). According toone embodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is a viable population of stem cells ofapical papilla. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is a viablepopulation of stem cells of apical papilla. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is a viable population of dental pulp stem cells.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is a viable population ofstem cells from exfoliated deciduous teeth. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is a viable population of periodontal ligament stem cells.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is a viable population ofdental follicle stem cells. Exemplary tissuegenic cells derived fromdental tissue are listed in Fong et al. (2005), J. Dent. Educ., 69(5):555-570, and Ulmer et al. (2010), Schweiz Monatsschr Zahnmed,120:860-872, the entire contents of which are incorporated herein byreference.

According to one embodiment, the at least one viable population oftissuegenic cells from dental tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into cells of an endodermal lineage. Exemplary targetlineages of tissuegenic cells derived from dental tissue are listed inUlmer et al. (2010), Schweiz Monatsschr Zahnmed, 120:860-872, the entirecontents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable of regeneratinga target tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from dental tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of regenerating a cartilagetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofregenerating a chorion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from dental tissue iscapable of regenerating a colon tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from dentaltissue is capable of regenerating a dental tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of regenerating a dermal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable of regeneratinga duodenal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofregenerating an endothelial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from dentaltissue is capable of regenerating a gastrointestinal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from dental tissue is capable of regenerating a growthplate tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is is capableof regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is is capable of regenerating an intestinalmucosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is is capableof regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is is capable of regenerating a kidneytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from dentaltissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable of regeneratinga meniscal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofregenerating a muscle tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from dental tissue iscapable of regenerating a nerve tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from dentaltissue is capable of regenerating an ovarian tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of regenerating a pancreatictissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofregenerating a parenchymal organ tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of regenerating a pericardial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from dental tissue is capable of regenerating a periostealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from dentaltissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of regenerating a spleen tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable of regeneratinga synovial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofregenerating a tendon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from dental tissue iscapable of regenerating a testes tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of regenerating a urological tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from dental tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into a colon cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of differentiating into a dental cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of differentiating into a duodenal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofdifferentiating into an endothelial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from dental tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from dentaltissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of differentiating into a meniscal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofdifferentiating into a muscle cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of differentiating into a nerve cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from dental tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from dentaltissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from dental tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from dental tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue is capable ofdifferentiating into a testes cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue is capable of differentiating into a urological celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from dental tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromdental tissue differentiates along a chondrogenic lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from dental tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from dental tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from dental tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from dental tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from dental tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Epithelial Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue comprises a viablegastrointestinal-derived stem cell population. Exemplary tissuegeniccells derived from gastrointestinal tissue are listed in U.S. PublishedApplication No. 2009/0269769, the entire contents of which areincorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from epithelial tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from epithelial tissue iscapable of differentiating into cells of an ectodermal lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofdifferentiating into cells of a mesodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into cellsof an endodermal lineage. Exemplary target lineages of tissuegenic cellsderived from epithelial tissue are listed in Ulmer et al. (2010),Schweiz Monatsschr Zahnmed, 120:860-872, the entire contents of whichare incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a dental tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a dermaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating a duodenal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating an endothelial tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating agastrointestinal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of regenerating a growth plate tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is is capable of regenerating anintervertebral disc tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from epithelialtissue is is capable of regenerating an intestinal mucosal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is is capable ofregenerating an intestinal serosal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromepithelial tissue is is capable of regenerating a kidney tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a nervetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from epithelial tissue iscapable of regenerating a pericardial tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a periostealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a spleentissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a testestissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofregenerating a urological tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of regenerating a vascular tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a cartilage cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into adental cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from epithelial tissue is capable of differentiating intoan epithelial cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from epithelialtissue is capable of differentiating into a gastrointestinal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from epithelial tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into akidney cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a ligament cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into aliver cell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into anerve cell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from epithelial tissue iscapable of differentiating into an ovarian cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from epithelialtissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from epithelial tissue is capable of differentiating intoa spleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into atendon cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from gastrointestinal tissue differentiatesalong an osteogenic lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from gastrointestinaltissue differentiates along an adipogenic lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from gastrointestinal tissue differentiates along a chondrogeniclineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from gastrointestinal tissue differentiatesalong a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from epithelial tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromepithelial tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from epithelial tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from epithelial tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from epithelial tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from epithelial tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from epithelial tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Tissuegenic Cells Derived from Fascial Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is selected from the groupconsisting of a viable pluripotent stem cell population, a viablemultipotent stem cell population, a viable progenitor cell population,and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells from fascial tissue secretes at least one growthinductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into cells ofa mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of differentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratinga target tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a fascial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating an amnion tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating an artery tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating a bone tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratinga cartilage tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a chorion tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a colon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating a dental tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratinga dermal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a duodenal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating an endothelial tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating an epithelialtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from fascial tissue is capable ofregenerating a gastrointestinal tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a growth plate tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating an intervertebraldisc tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating an intestinal mucosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating an intestinalserosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a kidney tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a ligament tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating a liver tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratinga lung tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratingan ovarian tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a pancreatic tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a parenchymal organ tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from fascial tissue is capable of regenerating apericardial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a periosteal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratinga spleen tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof regenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of regeneratinga urological tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of regenerating a vascular tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromfascial tissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a fascial cell lineage,a gastrointestinal cell lineage, a growth plate cell lineage, anintervertebral disc cell lineage, an intestinal mucosal cell lineage, anintestinal serosal cell lineage, a kidney cell lineage, a ligament celllineage, a liver cell lineage, a lung cell lineage, a meniscal celllineage, a muscle cell lineage, a nerve cell lineage, an ovarian celllineage, a pancreatic cell lineage, a parenchymal organ cell lineage, apericardial cell lineage, a periosteal cell lineage, a peritoneal celllineage, a skin cell lineage, a spleen cell lineage, a synovial celllineage, a tendon cell lineage, a testes cell lineage, a urological celllineage, a vascular cell lineage, a vein cell lineage, and a combinationthereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from fascial tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of differentiating into a fascial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into agastrointestinal cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from fascial tissueis capable of differentiating into a growth plate cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable ofdifferentiating into an intervertebral disc cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into anintestinal mucosal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of differentiating into an intestinal serosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from fascial tissue is capable ofdifferentiating into a kidney cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromfascial tissue is capable of differentiating into a ligament celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from fascial tissue is capable ofdifferentiating into a liver cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromfascial tissue is capable of differentiating into a lung cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable ofdifferentiating into a meniscal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into a musclecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into a nerve cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into anovarian cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of differentiating into a pancreatic cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into aparenchymal organ cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from fascialtissue is capable of differentiating into a pericardial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable ofdifferentiating into a periosteal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cells iscapable of differentiating into a peritoneal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into a skincell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into a spleen cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into asynovial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of differentiating into a tendon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into a testescell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue is capableof differentiating into a urological cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue is capable of differentiating into avascular cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from fascial tissue iscapable of differentiating into a vein cell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue can be reprogrammed to atleast one viable induced pluripotent stem cell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromfascial tissue differentiates along a chondrogenic lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from fascial tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from fascial tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from fascial tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from fascial tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Ligament Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue comprises a viableligament-derived mesenchymal stem cell population. Exemplary tissuegeniccells derived from ligament tissue are listed in Cheng et al. (2010),Tissue Engg. A, 16(7):2237-2253, the entire contents of which areincorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from ligament tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into cells ofa mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from ligament tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from ligament tissueis capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from ligament tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from ligament tissueis capable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromligament tissue is capable of regenerating a dermal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a duodenaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from ligament tissue is capable ofregenerating an endothelial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a gastrointestinal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from ligament tissue is capable of regenerating a growthplate tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is iscapable of regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is is capable of regenerating an intestinalmucosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is iscapable of regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is is capable of regenerating a kidneytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from ligament tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof regenerating a pericardial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived fromligament tissue is capable of regenerating a periosteal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a spleen tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofregenerating a urological tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of regenerating a vascular tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from ligament tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from ligament tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from ligamenttissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from ligament tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissue is capableof differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from ligament tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromligament tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from ligament tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from ligament tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from ligament tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from ligament tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from ligament tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Tissuegenic Cells Derived from Lung Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue comprises a viablelung-derived stem cell population. Exemplary tissuegenic cells derivedfrom lung tissue are disclosed in Kajstura et al. (2011), N. Engl. J.Med., 364(19):1795-1806, the entire contents of which are incorporatedherein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from lung tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto cells of at least one embryonic lineage. According to oneembodiment, the embryonic lineage is selected from the group consistingof an ectodermal lineage, a mesodermal lineage and an endodermallineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from lung tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating atarget tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating anadipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of regenerating a cartilage tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating achorion tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissue iscapable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of regenerating a dermal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of regenerating a duodenal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating anendothelial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating a gastrointestinal tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from lungtissue is capable of regenerating a growth plate tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is is capable of regenerating an intervertebraldisc tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is is capableof regenerating an intestinal mucosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is is capable of regenerating an intestinalserosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is is capableof regenerating a kidney tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of regenerating a ligament tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of regenerating a liver tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating alung tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating anovarian tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating a pancreatic tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of regenerating a parenchymal organ tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from lung tissue is capable of regenerating a pericardialtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from lung tissue is capable of regeneratinga periosteal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from lung tissue iscapable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating aspleen tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of regenerating aurological tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofregenerating a vascular tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto a target tissue cell lineage. According to one embodiment, thetarget tissue cell lineage is selected from the group consisting of anadipose cell lineage, an amnion cell lineage, an artery cell lineage, abone cell lineage, a cartilage cell lineage, a dental cell lineage, adermal cell lineage, a duodenal cell lineage, an endothelial lineage, anepithelial cell lineage, a gastrointestinal cell lineage, a growth platecell lineage, an intervertebral disc cell lineage, an intestinal mucosalcell lineage, an intestinal serosal cell lineage, a kidney cell lineage,a ligament cell lineage, a liver cell lineage, a lung cell lineage, ameniscal cell lineage, a muscle cell lineage, a nerve cell lineage, anovarian cell lineage, a pancreatic cell lineage, a parenchymal organcell lineage, a pericardial cell lineage, a periosteal cell lineage, aperitoneal cell lineage, a skin cell lineage, a spleen cell lineage, asynovial cell lineage, a tendon cell lineage, a testes cell lineage, aurological cell lineage, a vascular cell lineage, a vein cell lineage,and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto an adipose cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissue iscapable of differentiating into an amnion cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into an arterycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into a bone cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of differentiating into a cartilage cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto a chorion cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of differentiating into a duodenal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto an endothelial cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of differentiating into an epithelial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto a gastrointestinal cell lineage. According to one embodiment, theat least one viable population of tissuegenic cells derived from lungtissue is capable of differentiating into a growth plate cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto an intervertebral disc cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of differentiating into an intestinal mucosalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into a liver celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from lung tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of differentiating into a meniscal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto a muscle cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissue iscapable of differentiating into a nerve cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into an ovariancell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into a pancreatic cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into aparenchymal organ cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from lungtissue is capable of differentiating into a pericardial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto a periosteal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells is capable ofdifferentiating into a peritoneal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into a skin celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from lung tissue is capable ofdifferentiating into a spleen cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromlung tissue is capable of differentiating into a synovial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of differentiatinginto a tendon cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into a urologicalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue is capable of differentiating into a vein celllineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue differentiatesalong an adipogenic lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from lung tissuedifferentiates along a chondrogenic lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from lung tissue is capable of migrating fromor to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from lung tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from lung tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from lung tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Mammary Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue comprises a viablemammary-derived stem cell population. Exemplary tissuegenic cellsderived from mammary tissue are listed in LaBarge, 2007, Stem Cell Rev.,3(2): 137-146, the entire contents of which are incorporated herein byreference.

According to one embodiment, the at least one viable population oftissuegenic cells from mammary tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into cells ofa mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into cells of an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable of regeneratinga target tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof regenerating an amnion tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from mammarytissue is capable of regenerating an artery tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating a bone tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable of regeneratinga cartilage tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof regenerating a chorion tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from mammarytissue is capable of regenerating a colon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating a dental tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable of regeneratinga dermal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof regenerating a duodenal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from mammarytissue is capable of regenerating an endothelial tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating agastrointestinal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of regenerating a growth plate tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is is capable of regenerating anintervertebral disc tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from mammary tissueis is capable of regenerating an intestinal mucosal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is is capable of regenerating an intestinalserosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is iscapable of regenerating a kidney tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frommammary tissue is capable of regenerating a ligament tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from mammary tissue is capable of regenerating a livertissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from mammary tissue is capable ofregenerating a lung tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from mammary tissueis capable of regenerating a meniscal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating a muscle tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable of regeneratinga nerve tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof regenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from mammarytissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof regenerating a pericardial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from mammarytissue is capable of regenerating a periosteal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating a peritonealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from mammary tissue is capable ofregenerating a skin tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from mammary tissueis capable of regenerating a spleen tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frommammary tissue is capable of regenerating a synovial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from mammary tissue is capable of regenerating a tendontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from mammary tissue is capable ofregenerating a testes tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from mammary tissueis capable of regenerating a urological tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from mammary tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from mammary tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from mammarytissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from mammary tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from mammarytissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from mammary tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue is capableof differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from mammary tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommammary tissue differentiates along a chondrogenic lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from mammary tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from mammary tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from mammary tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Muscle Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is selected from the groupconsisting of a viable cardiac muscle stem cell population, a viableskeletal muscle stem cell population and a viable smooth muscle stemcell population. According to one embodiment, the at least viablepopulation of tissuegenic cells derived from muscle tissue comprises aviable cardiac muscle stem cell population. According to one embodiment,the at least viable population of tissuegenic cells derived from muscletissue comprises a viable skeletal muscle stem cell population.According to one embodiment, the at least viable population oftissuegenic cells derived from muscle tissue comprises a viable smoothmuscle stem cell population.

According to one embodiment, the at least one viable population oftissuegenic cells from muscle tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into cells of an endodermal lineage. Exemplary targetlineages of tissuegenic cells derived from muscle tissue are listed inXu et al. (2010), Cell Tissue Res., 340: 549-567, the entire contents ofwhich are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable of regeneratinga target tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from muscle tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of regenerating a cartilagetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofregenerating a chorion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from muscle tissue iscapable of regenerating a colon tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from muscletissue is capable of regenerating a dental tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of regenerating a dermal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable of regeneratinga duodenal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofregenerating an endothelial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from muscletissue is capable of regenerating a gastrointestinal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from muscle tissue is capable of regenerating a growthplate tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is is capableof regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is is capable of regenerating an intestinalmucosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is is capableof regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is is capable of regenerating a kidneytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from muscletissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable of regeneratinga meniscal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofregenerating a muscle tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from muscle tissue iscapable of regenerating a nerve tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from muscletissue is capable of regenerating an ovarian tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of regenerating a pancreatictissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofregenerating a parenchymal organ tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of regenerating a pericardial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from muscle tissue is capable of regenerating a periostealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from muscletissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of regenerating a spleen tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable of regeneratinga synovial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofregenerating a tendon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from muscle tissue iscapable of regenerating a testes tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of regenerating a urological tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from muscle tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from mammary tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a colon cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of differentiating into a dental cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of differentiating into a duodenal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofdifferentiating into an endothelial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from muscle tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from muscletissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of differentiating into a meniscal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a muscle cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of differentiating into a nerve cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from muscle tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from muscletissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from muscle tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from muscle tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a testes cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue is capable of differentiating into a urological celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from muscle tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived frommuscle tissue differentiates along a chondrogenic lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from muscle tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from muscle tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from muscle tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from muscle tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Nerve Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue comprises a viable neuralstem cell population. Exemplary tissuegenic cells derived from nervetissue are listed in Alvarez-Buylla and Lim (2004), Neuron, 41: 683-686,the entire contents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from nerve tissue secretes at least onegrowth-inductive factor. Exemplary growth factors secreted bytissuegenic cells derived from nerve tissue are listed in Alvarez-Buyllaand Lim (2004), Neuron, 41: 683-686, the entire contents of which areincorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into cells of an endodermal lineage. Exemplary targetlineages of tissuegenic cells derived from nerve tissue are listed in Xuet al. (2010), Cell Tissue Res., 340: 549-567, the entire contents ofwhich are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regenerating atarget tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from nerve tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a cartilage tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regenerating achorion tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from nerve tissue iscapable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of regenerating a dermal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a duodenal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regeneratingan endothelial tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from nerve tissue iscapable of regenerating a gastrointestinal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a growth platetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from nerve tissue is is capable ofregenerating an intervertebral disc tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is is capable of regenerating an intestinal mucosal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is is capable ofregenerating an intestinal serosal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is is capable of regenerating a kidney tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a ligament tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regenerating aliver tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofregenerating a lung tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from nerve tissue iscapable of regenerating a meniscal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of regenerating a muscle tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regeneratingan ovarian tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofregenerating a pancreatic tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from nervetissue is capable of regenerating a parenchymal organ tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from nerve tissue is capable of regenerating a pericardialtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from nerve tissue is capable ofregenerating a periosteal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from nervetissue is capable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regenerating aspleen tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from nervetissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of regenerating aurological tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofregenerating a vascular tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from nervetissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into a bone celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from nerve tissue is capable ofdifferentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into a colon cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of differentiating into a dental cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of differentiating into a duodenal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into an endothelial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from nerve tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from nervetissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from nerve tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of differentiating into a meniscal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into a muscle cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of differentiating into a nerve cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from nerve tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from nervetissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from nerve tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from nerve tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue is capable ofdifferentiating into a testes cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromnerve tissue is capable of differentiating into a urological celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from nerve tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of differentiating into a vein celllineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue differentiatesalong an adipogenic lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from nerve tissuedifferentiates along a chondrogenic lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from nerve tissue is capable of migrating fromor to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from nerve tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from nerve tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Periosteal Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue comprises a viableperiosteum-derived stem cell population. Exemplary tissuegenic cellsderived from periosteal tissue are listed in Zhang et al., 2005, J.Musculoskelet. Neuronal. Interact. 5(4): 360-362, the entire contents ofwhich are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from periosteal tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from periosteal tissue iscapable of differentiating into cells of an ectodermal lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofdifferentiating into cells of a mesodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into cellsof an endodermal lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a dental tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a dermaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating a duodenal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating an endothelial tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating agastrointestinal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of regenerating a growth plate tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is is capable of regenerating anintervertebral disc tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from periostealtissue is is capable of regenerating an intestinal mucosal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is is capable ofregenerating an intestinal serosal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromperiosteal tissue is is capable of regenerating a kidney tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a nervetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from periosteal tissue iscapable of regenerating a pericardial tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a periostealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a spleentissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a testestissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofregenerating a urological tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of regenerating a vascular tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a cartilage cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into adental cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from periosteal tissue is capable of differentiating intoan epithelial cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from periostealtissue is capable of differentiating into a gastrointestinal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from periosteal tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into akidney cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a ligament cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into aliver cell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into anerve cell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from periosteal tissue iscapable of differentiating into an ovarian cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from periostealtissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from periosteal tissue is capable of differentiating intoa spleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into atendon cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from periosteal tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromperiosteal tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from periosteal tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from periosteal tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from periosteal tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from periosteal tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from periosteal tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Tissuegenic Cells Derived from Placental Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is selected from thegroup consisting of a viable population of amniotic stem cells, a viablepopulation of pluripotent stem cells, a viable population ofamnion-derived mesenchymal stem cells, and a viable population ofchorion-derived stem cells. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis a viable pluripotent stem cell population. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is a viable amnion-derived mesenchymalstem cell population. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental is aviable chorion-derived mesenchymal stem cell population. Exemplaryamnion-derived and chorion-derived stem cells are listed in Wei J. etal., Cell Transplant, 2003, 12: 545-552; Wolbank, S. et al., Tissue Eng,2007, 13: 1173-1183; Alviano, F. et al., BMC Dev Biol, 2007, 7: 11;Zhao, P. et al, Transplantation, 2005, 79: 528-535, the entire contentsof which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from placental tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into cellsof a mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into cells of an endodermal lineage.Exemplary target lineages generated from tissuegenic cells derived fromplacental tissue are listed in Int′Anker, P. et al., Stem Cells, 2004,22: 1338-1345; Portmann-Lanz, C. et al, Am J Obstet Gynecol, 2006, 194:664-673; Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Soncini,M. et al., J Tissue Eng Regen Med, 2007, 1:296-305; Alviano, F., BMC DevBiol, 2007, 7: 11, the entire contents of which are incorporated hereinby reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from placentaltissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromplacental tissue is capable of regenerating a dermal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from placental tissue is capable of regenerating aduodenal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from placental tissue is capableof regenerating an endothelial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived fromplacental tissue is capable of regenerating a gastrointestinal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofregenerating a growth plate tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from placentaltissue is is capable of regenerating an intervertebral disc tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is is capable ofregenerating an intestinal mucosal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromplacental tissue is is capable of regenerating an intestinal serosaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is is capable ofregenerating a kidney tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating a ligament tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from nerve tissue is capable of regenerating a liver tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofregenerating a lung tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating a meniscal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating a muscletissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofregenerating a nerve tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating an ovarian tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating a pancreatictissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofregenerating a parenchymal organ tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromplacental tissue is capable of regenerating a pericardial tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofregenerating a periosteal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from placentaltissue is capable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofregenerating a spleen tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating a synovial tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating a tendontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofregenerating a testes tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of regenerating a urological tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from placental tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into adental cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from placental tissue is capable of differentiating intoan epithelial cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from placental tissueis capable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from placentaltissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from placental tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into akidney cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a ligament cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from placental tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from placental tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from placentaltissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from placental tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into atendon cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from placental tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from placental tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromplacental tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from placental tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from placental tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from placental tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from placental tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from placental tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Tissuegenic Cells Derived from Skin Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is selected from the groupconsisting of a viable population of epidermal stem cells ofinterfollicular epidermis, a viable population of bulge stem cells, aviable population of epidermal stem cells of a hair follicle, dermisderived muiltipotent cells, dermis derived progenitor cells and dermisderived fibrocytes. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue comprises aviable population of epidermal stem cells of interfollicular epidermis.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue comprises a viable populationof bulge stem cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from skin tissuecomprises a viable population of epidermal stem cells of a hairfollicle. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue comprises aviable population of dermis derived muiltipotent cells. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue comprises a viable population of dermis derivedprogenitor cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue comprises aviable population of dermis derived fibrocytes. Exemplary tissuegeniccells derived from skin tissue are listed in Zouboulis et al., 2008,Exp. Gerontol. 43: 986-997; Blanpain, 2010, Nature, 464: 686-687, theentire contents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from skin tissue secretes at least onegrowth-inductive factor. Exemplary growth-inductive factors secreted bytissuegenic cells derived from skin tissue are disclosed in Blanpain andFuchs, 2009, Nat. Rev. Mol. Cell. Biol., 10(3): 207-217, the entirecontents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto cells of at least one embryonic lineage. According to oneembodiment, the embryonic lineage is selected from the group consistingof an ectodermal lineage, a mesodermal lineage and an endodermallineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from skin tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into cells of an endodermal lineage. Exemplary targetlineages generated from tissuegenic cells derived from skin tissue arelisted in Int′Anker, P. et al., Stem Cells, 2004, 22: 1338-1345;Portmann-Lanz, C. et al, Am J Obstet Gynecol, 2006, 194: 664-673;Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Soncini, M. et al.,J Tissue Eng Regen Med, 2007, 1:296-305; Alviano, F., BMC Dev Biol,2007, 7: 11, the entire contents of which are incorporated herein byreference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating atarget tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating anadipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of regenerating a cartilage tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating achorion tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissue iscapable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of regenerating a dermal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of regenerating a duodenal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating anendothelial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating a gastrointestinal tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from skintissue is capable of regenerating a growth plate tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is is capable of regenerating an intervertebraldisc tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is is capableof regenerating an intestinal mucosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is is capable of regenerating an intestinalserosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is is capableof regenerating a kidney tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of regenerating a ligament tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of regenerating a liver tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating alung tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating anovarian tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating a pancreatic tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of regenerating a parenchymal organ tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from skin tissue is capable of regenerating a pericardialtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from skin tissue is capable of regeneratinga periosteal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from skin tissue iscapable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of regenerating a skin tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating aspleen tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of regenerating aurological tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofregenerating a vascular tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto a target tissue cell lineage. According to one embodiment, thetarget tissue cell lineage is selected from the group consisting of anadipose cell lineage, an amnion cell lineage, an artery cell lineage, abone cell lineage, a cartilage cell lineage, a dental cell lineage, adermal cell lineage, a duodenal cell lineage, an endothelial lineage, anepithelial cell lineage, a gastrointestinal cell lineage, a growth platecell lineage, an intervertebral disc cell lineage, an intestinal mucosalcell lineage, an intestinal serosal cell lineage, a kidney cell lineage,a ligament cell lineage, a liver cell lineage, a lung cell lineage, ameniscal cell lineage, a muscle cell lineage, a nerve cell lineage, anovarian cell lineage, a pancreatic cell lineage, a parenchymal organcell lineage, a pericardial cell lineage, a periosteal cell lineage, aperitoneal cell lineage, a skin cell lineage, a spleen cell lineage, asynovial cell lineage, a tendon cell lineage, a testes cell lineage, aurological cell lineage, a vascular cell lineage, a vein cell lineage,and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto an adipose cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissue iscapable of differentiating into an amnion cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into an arterycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into a bone cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of differentiating into a cartilage cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto a chorion cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of differentiating into a duodenal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto an endothelial cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of differentiating into an epithelial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto a gastrointestinal cell lineage. According to one embodiment, theat least one viable population of tissuegenic cells derived from skintissue is capable of differentiating into a growth plate cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto an intervertebral disc cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of differentiating into an intestinal mucosalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into a liver celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from skin tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of differentiating into a meniscal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto a muscle cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissue iscapable of differentiating into a nerve cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into an ovariancell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into a pancreatic cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into aparenchymal organ cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from skintissue is capable of differentiating into a pericardial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto a periosteal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells is capable ofdifferentiating into a peritoneal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into a skin celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from skin tissue is capable ofdifferentiating into a spleen cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromskin tissue is capable of differentiating into a synovial cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of differentiatinginto a tendon cell lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissue iscapable of differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into a urologicalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue is capable of differentiating into a vein celllineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue differentiatesalong an adipogenic lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from skin tissuedifferentiates along a chondrogenic lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from skin tissue is capable of migrating fromor to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from skin tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from skin tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from skin tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Synovial Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is selected from thegroup consisting of a viable population of synovial-derived stem cellsand synovial-derived progenitor cells. According to one embodiment, theat least one viable population of tissuegenic cells derived fromsynovial tissue comprises a viable population of synovial-derived stemcells. According to one embodiment, the at least one viable populationof tissuegenic cells derived from synovial tissue comprises a viablepopulation of synovial-derived progenitor cells. Exemplary tissuegeniccells derived from synovial tissue are disclosed in Kurth et al.,Arthritis Rheum., 2011, 63(5): 1289-1300, the entire contents of whichare incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from synovial tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into cells ofa mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into cells of an endodermal lineage.Exemplary target lineages generated from tissuegenic cells derived fromsynovial tissue are disclosed in Koga et al., 2008, Cell Tissue Res.,333: 207-215, Miyamoto et al., 2010, Arthritis Res. Ther., 12: R206-218;and Lee et al., 2010, Tissue Engg. A, 16(1): 317-325, the entirecontents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from synovial tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from synovial tissueis capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from synovial tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from synovial tissueis capable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromsynovial tissue is capable of regenerating a dermal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a duodenaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from synovial tissue is capable ofregenerating an endothelial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a gastrointestinal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from synovial tissue is capable of regenerating a growthplate tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is iscapable of regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is is capable of regenerating an intestinalmucosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is iscapable of regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is is capable of regenerating a kidneytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from synovial tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof regenerating a pericardial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived fromsynovial tissue is capable of regenerating a periosteal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a spleen tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofregenerating a urological tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of regenerating a vascular tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from synovial tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from synovial tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from synovialtissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from synovial tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissue is capableof differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from synovial tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromsynovial tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from synovial tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from synovial tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from synovial tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from synovial tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from synovial tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Tissuegenic Cells Derived from Tendon Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is selected from the groupconsisting of a viable population of tendon-derived stem cells andtendon derived progenitor cells. According to one embodiment, the atleast one viable population of tissuegenic cells derived from tendontissue comprises a viable population of tendon-derived stem cells.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue comprises a viablepopulation of tendon-derived progenitor cells. Exemplary tissuegeniccells derived from synovial tissue are disclosed in Bi et al., 2007,Nat. Med., 13(10): 1219-1227, the entire contents of which areincorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from tendon tissue secretes at least onegrowth-inductive factor.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into cells of amesodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into cells of an endodermal lineage. Exemplary targetlineages generated from tissuegenic cells derived from tendon tissue aredisclosed in Koga et al., 2008, Cell Tissue Res., 333: 207-215, Miyamotoet al., 2010, Arthritis Res. Ther., 12: R206-218; and Lee et al., 2010,Tissue Engg. A, 16(1): 317-325, the entire contents of which areincorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable of regeneratinga target tissue. According to one embodiment, the target tissue isselected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endothelial tissue, an epithelial tissue, a gastrointestinaltissue, a growth plate tissue, an intervertebral disc tissue, anintestinal mucosal tissue, an intestinal serosal tissue, a kidneytissue, a ligament tissue, a liver tissue, a lung tissue, a meniscaltissue, a muscle tissue, a nerve tissue, an ovarian tissue, a pancreatictissue, a parenchymal organ tissue, a pericardial tissue, a periostealtissue, a peritoneal tissue, a skin tissue, a spleen tissue, a synovialtissue, a tendon tissue, a testes tissue, a urological tissue, avascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable of regeneratingan adipose tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofregenerating an amnion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from tendon tissue iscapable of regenerating an artery tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of regenerating a bone tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of regenerating a cartilagetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofregenerating a chorion tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from tendon tissue iscapable of regenerating a colon tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from tendontissue is capable of regenerating a dental tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of regenerating a dermal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable of regeneratinga duodenal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofregenerating an endothelial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from tendontissue is capable of regenerating a gastrointestinal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from tendon tissue is capable of regenerating a growthplate tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is is capableof regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is is capable of regenerating an intestinalmucosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is is capableof regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is is capable of regenerating a kidneytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from tendontissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable of regeneratinga meniscal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofregenerating a muscle tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from tendon tissue iscapable of regenerating a nerve tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived from tendontissue is capable of regenerating an ovarian tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of regenerating a pancreatictissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofregenerating a parenchymal organ tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of regenerating a pericardial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from tendon tissue is capable of regenerating a periostealtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from tendontissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of regenerating a spleen tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable of regeneratinga synovial tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofregenerating a tendon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from tendon tissue iscapable of regenerating a testes tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of regenerating a urological tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from tendon tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into an amnioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into a chorioncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a colon cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of differentiating into a dental cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into a dermal cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of differentiating into a duodenal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofdifferentiating into an endothelial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from tendon tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from tendontissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a lung cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of differentiating into a meniscal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a muscle cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of differentiating into a nerve cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from tendon tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from tendontissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from tendon tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from tendon tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a testes cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue is capable of differentiating into a urological celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from tendon tissue is capable ofdifferentiating into a vascular cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromtendon tissue differentiates along a chondrogenic lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue differentiates along a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from tendon tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from tendon tissue comprises a viable nonexpanded population oftissuegenic cells. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from tendon tissue comprises aviable expanded population of tissuegenic cells. According to someembodiments, the at least one viable population of tissuegenic cellsderived from tendon tissue adherent to and resident in the endogenousmilieu of the growth conductive matrix is immune privileged. The term“immune privileged” as used herein refers to the characteristic oftissuegenic cells by which there is no induction of an immune responseupon transplantation of such cells.

Tissuegenic Cells Derived from Umbilical Cord Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is selected fromthe group consisting of a viable population of umbilical cord-derivedhematopoietic stem cells (UC-HS), a viable population of umbilicalcord-derived mesenchyma stem cells (UC-MS), and a viable population ofumbilical cord-derived Wharton's Jelly stem cells (UC-MM). According toone embodiment, the at least one viable population of tissuegenic cellsderived from umbilical cordtissue comprises a viable population ofumbilical cord-derived hematopoietic stem cells (UC-HS). According toone embodiment, the at least one viable population of tissuegenic cellsderived from umbilical cordtissue comprises a viable population ofumbilical cord-derived mesenchyma stem cells (UC-MS). According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cordtissue comprises a viable population ofumbilical cord-derived Wharton's Jelly stem cells (UC-MM). Exemplarytissuegenic cells derived from umbilical cord tissue are disclosed inMunn, D. et al., Science, 1998, 281: 1191-1193; Munn, D. et al., J ExpMed, 1999, 189: 1363-1372, the entire contents of which are incorporatedherein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from umbilical cord tissue secretes at least onegrowth-inductive factor. Exemplary growth-inductive factors secreted bytissuegenic cells derived from umbilical cord tissue are disclosed inZhang, X et al., Biochem Biophys Res Commun, 2006, 351: 853-859, theentire contents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from umbilical cord tissue iscapable of differentiating into cells of an ectodermal lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into cells of a mesodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating intocells of an endodermal lineage. Exemplary target lineages generated fromtissuegenic cells derived from umbilical cord tissue are disclosed inKoga et al., 2008, Cell Tissue Res., 333: 207-215, Miyamoto et al.,2010, Arthritis Res. Ther., 12: R206-218; and Lee et al., 2010, TissueEngg. A, 16(1): 317-325, the entire contents of which are incorporatedherein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of regenerating an amnion tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from umbilical cordtissue is capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from umbilical cordtissue is capable of regenerating a dental tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a dermaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a duodenal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of regenerating an endothelial tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from umbilical cord tissue is capable of regenerating agastrointestinal tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of regenerating a growth plate tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is is capable of regenerating anintervertebral disc tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from umbilical cordtissue is is capable of regenerating an intestinal mucosal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is is capable ofregenerating an intestinal serosal tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromumbilical cord tissue is is capable of regenerating a kidney tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a lungtissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a nervetissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of regenerating a pancreatic tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating aparenchymal organ tissue. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of regenerating a pericardial tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating aperiosteal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from umbilical cord tissue iscapable of regenerating a peritoneal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a skintissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a spleen tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from umbilical cordtissue is capable of regenerating a synovial tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a tendontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a testes tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from umbilical cordtissue is capable of regenerating a urological tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of regenerating a vasculartissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofregenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of differentiating into an artery cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a bone cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromumbilical cord tissue is capable of differentiating into a cartilagecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from umbilical cord tissue iscapable of differentiating into a chorion cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into acolon cell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from umbilical cord tissue iscapable of differentiating into a dental cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into adermal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of differentiating into a duodenal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into an endothelial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of differentiating into a gastrointestinal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of differentiating into an intestinal mucosalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from umbilical cord tissue iscapable of differentiating into an intestinal serosal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a kidney cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromumbilical cord tissue is capable of differentiating into a ligament celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a liver cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromumbilical cord tissue is capable of differentiating into a lung celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a meniscal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into amuscle cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of differentiating into a nerve cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of differentiating into a parenchymal organ celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue is capable of differentiating into a skin cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a spleen cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromumbilical cord tissue is capable of differentiating into a synovial celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a tendon cell lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromumbilical cord tissue is capable of differentiating into a testes celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue is capable ofdifferentiating into a urological cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue is capable of differentiating into avascular cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from umbilical cordtissue is capable of differentiating into a vein cell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue differentiatesalong an osteogenic lineage. According to one embodiment, the at leastone viable population of tissuegenic cells derived from umbilical cordtissue differentiates along an adipogenic lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue differentiates along a chondrogeniclineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from umbilical cord tissue differentiatesalong a neurogenic lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue is capable ofmigrating from or to the at least one growth-conductive matrix.According to one embodiment, the at least one viable population oftissuegenic cells derived from umbilical cord tissue comprises a viablenonexpanded population of tissuegenic cells. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from umbilical cord tissue comprises a viable expandedpopulation of tissuegenic cells. According to some embodiments, the atleast one viable population of tissuegenic cells derived from umbilicalcord tissue adherent to and resident in the endogenous milieu of thegrowth conductive matrix is immune privileged. The term “immuneprivileged” as used herein refers to the characteristic of tissuegeniccells by which there is no induction of an immune response upontransplantation of such cells.

Tissuegenic Cells Derived from Vascular Tissue

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is selected from thegroup consisting of a viable population of progenitor cells and a viablepopulation of stem cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissuecomprises a viable population of progenitor cells. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue comprises a viable population of stemcells. Exemplary tissuegenic cells derived from vascular tissue aredisclosed in Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509, theentire contents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells from vascular tissue secretes at least onegrowth-inductive factor. Exemplary growth-inductive factors secreted bytissuegenic cells derived from vascular tissue are disclosed in Tilki etal., 2009, Trends Mol. Med. 15(11): 501-509, the entire contents ofwhich are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofdifferentiating into cells of at least one embryonic lineage. Accordingto one embodiment, the embryonic lineage is selected from the groupconsisting of an ectodermal lineage, a mesodermal lineage and anendodermal lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into cells of an ectodermal lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into cells ofa mesodermal lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into cells of an endodermal lineage.Exemplary target lineages generated from tissuegenic cells derived fromvascular tissue are disclosed in Koga et al., 2008, Cell Tissue Res.,333: 207-215, Miyamoto et al., 2010, Arthritis Res. Ther., 12: R206-218;and Lee et al., 2010, Tissue Engg. A, 16(1): 317-325, the entirecontents of which are incorporated herein by reference.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating a target tissue. According to one embodiment, the targettissue is selected from the group consisting of an adipose tissue, anamnion tissue, an artery tissue, a bone tissue, a cartilage tissue, achorion tissue, a colon tissue, a dental tissue, a dermal tissue, aduodenal tissue, an endothelial tissue, an epithelial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, ameniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, apancreatic tissue, a parenchymal organ tissue, a pericardial tissue, aperiosteal tissue, a peritoneal tissue, a skin tissue, a spleen tissue,a synovial tissue, a tendon tissue, a testes tissue, a urologicaltissue, a vascular tissue, a vein tissue, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating an adipose tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating an amnion tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating an arterytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from vascular tissue is capable ofregenerating a bone tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from vascular tissueis capable of regenerating a cartilage tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a choriontissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from vascular tissue is capable ofregenerating a colon tissue. According to one embodiment, the at leastone viable population of tissuegenic cells derived from vascular tissueis capable of regenerating a dental tissue. According to one embodiment,the at least one viable population of tissuegenic cells derived fromvascular tissue is capable of regenerating a dermal tissue. According toone embodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a duodenaltissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from vascular tissue is capable ofregenerating an endothelial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a gastrointestinal tissue. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from vascular tissue is capable of regenerating a growthplate tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is iscapable of regenerating an intervertebral disc tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is is capable of regenerating an intestinalmucosal tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is iscapable of regenerating an intestinal serosal tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is is capable of regenerating a kidneytissue. According to one embodiment, the at least one viable populationof tissuegenic cells derived from vascular tissue is capable ofregenerating a ligament tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a liver tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a lung tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating a meniscal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a muscle tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a nerve tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating an ovarian tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a pancreatic tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a parenchymalorgan tissue. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof regenerating a pericardial tissue. According to one embodiment, theat least one viable population of tissuegenic cells derived fromvascular tissue is capable of regenerating a periosteal tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating a peritoneal tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a skin tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a spleen tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating a synovial tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a tendon tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a testes tissue.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofregenerating a urological tissue. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of regenerating a vascular tissue. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of regenerating a vein tissue.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofdifferentiating into a target tissue cell lineage. According to oneembodiment, the target tissue cell lineage is selected from the groupconsisting of an adipose cell lineage, an amnion cell lineage, an arterycell lineage, a bone cell lineage, a cartilage cell lineage, a dentalcell lineage, a dermal cell lineage, a duodenal cell lineage, anendothelial lineage, an epithelial cell lineage, a gastrointestinal celllineage, a growth plate cell lineage, an intervertebral disc celllineage, an intestinal mucosal cell lineage, an intestinal serosal celllineage, a kidney cell lineage, a ligament cell lineage, a liver celllineage, a lung cell lineage, a meniscal cell lineage, a muscle celllineage, a nerve cell lineage, an ovarian cell lineage, a pancreaticcell lineage, a parenchymal organ cell lineage, a pericardial celllineage, a periosteal cell lineage, a peritoneal cell lineage, a skincell lineage, a spleen cell lineage, a synovial cell lineage, a tendoncell lineage, a testes cell lineage, a urological cell lineage, avascular cell lineage, a vein cell lineage, and a combination thereof.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofdifferentiating into an adipose cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into anamnion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into an artery cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a bonecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into a cartilage cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into achorion cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into a colon cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a dentalcell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into a dermal cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into aduodenal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into an endothelial cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from vascular tissue is capable of differentiating into anepithelial cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into a gastrointestinal cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofdifferentiating into a growth plate cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into anintervertebral disc cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of differentiating into an intestinal mucosal celllineage. According to one embodiment, the at least one viable populationof tissuegenic cells derived from vascular tissue is capable ofdifferentiating into an intestinal serosal cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a kidneycell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into a ligament cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a livercell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into a lung cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into ameniscal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into a muscle cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a nervecell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into an ovarian cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into apancreatic cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into a parenchymal organ cell lineage.According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable ofdifferentiating into a pericardial cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into aperiosteal cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells is capable of differentiatinginto a peritoneal cell lineage. According to one embodiment, the atleast one viable population of tissuegenic cells derived from vasculartissue is capable of differentiating into a skin cell lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from vascular tissue is capable of differentiating into aspleen cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into a synovial cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a tendoncell lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissue is capableof differentiating into a testes cell lineage. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into aurological cell lineage. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissue iscapable of differentiating into a vascular cell lineage. According toone embodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue is capable of differentiating into a veincell lineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue can bereprogrammedreprogrammed to at least one viable induced pluripotent stemcell (iPSC) population.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue differentiates along anosteogenic lineage. According to one embodiment, the at least one viablepopulation of tissuegenic cells derived from vascular tissuedifferentiates along an adipogenic lineage. According to one embodiment,the at least one viable population of tissuegenic cells derived fromvascular tissue differentiates along a chondrogenic lineage. Accordingto one embodiment, the at least one viable population of tissuegeniccells derived from vascular tissue differentiates along a neurogeniclineage.

According to one embodiment, the at least one viable population oftissuegenic cells derived from vascular tissue is capable of migratingfrom or to the at least one growth-conductive matrix. According to oneembodiment, the at least one viable population of tissuegenic cellsderived from vascular tissue comprises a viable nonexpanded populationof tissuegenic cells. According to one embodiment, the at least oneviable population of tissuegenic cells derived from vascular tissuecomprises a viable expanded population of tissuegenic cells. Accordingto some embodiments, the at least one viable population of tissuegeniccells derived from vascular tissue adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

Frequency of Tissuegenic Cells

According to some embodiments, the at least one viable population oftissuegenic cells comprise a relative frequency substantially similar tothe total cell population of the growth-conductive matrix. According tosome embodiments, the at least one viable population of tissuegeniccells comprise a relative frequency substantially higher than the totalcell population of the growth-conductive matrix.

According to some embodiments, the at least one viable population oftissuegenic cells comprise at least about 10% to at least about 95% ofthe total cell population of the growth-conductive matrix. According tosome embodiments, the at least one viable population of tissuegeniccells comprise at least about 10% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the viablepopulation of tissuegenic cells comprise at least about 15% of the totalcell population of the growth-conductive matrix. According to someembodiments, the at least one viable population of tissuegenic cellscomprise at least about 20% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 25%of the total cell population of the growth-conductive matrix. Accordingto some embodiments, the viable population of tissuegenic cells compriseat least about 30% of the total cell population of the growth-conductivematrix. According to some embodiments, the viable population oftissuegenic cells comprise at least about 35% of the total cellpopulation of the growth-conductive matrix. According to someembodiments, the at least one viable population of tissuegenic cellscomprise at least about 40% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 45%of the total cell population of the growth-conductive matrix. Accordingto some embodiments, the at least one viable population of tissuegeniccells comprise at least about 50% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 55%of the total cell population of the growth-conductive matrix. Accordingto some embodiments, the at least one viable population of tissuegeniccells comprise at least about 60% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 65%of the total cell population of the growth-conductive matrix. Accordingto some embodiments, the at least one viable population of tissuegeniccells comprise at least about 70% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 75%of the total cell population of the growth-conductive matrix. Accordingto some embodiments, the at least one viable population of tissuegeniccells comprise at least about 80% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 85%of the total cell population of the growth-conductive matrix. Accordingto some embodiments, the at least one viable population of tissuegeniccells comprise at least about 90% of the total cell population of thegrowth-conductive matrix. According to some embodiments, the at leastone viable population of tissuegenic cells comprise at least about 95%of the total cell population of the growth-conductive matrix.

According to some embodiments, the at least one viable population oftissuegenic cells comprise at least about at least about 10,000tissuegenic cells per cc of implant. According to some embodiments, theat least one viable population of tissuegenic cells comprise at leastabout at least about 20,000 tissuegenic cells per cc of implant.According to some embodiments, the at least one viable population oftissuegenic cells comprise at least about at least about 30,000tissuegenic cells per cc of implant. According to some embodiments, theat least one viable population of tissuegenic cells comprise at leastabout at least about 40,000 tissuegenic cells per cc of implant.According to some embodiments, the at least one viable population oftissuegenic cells comprise at least about at least about 50,000tissuegenic cells per cc of implant.

Growth-Inductive Component

According to some embodiments, the implant further comprises at leastone growth-inductive component. According to some such embodiments, thegrowth-inductive component is at least one cytokine According to somesuch embodiments, the at least one growth-inductive component comprisesat least one growth factor. According to some such embodiments, the atleast one growth factor is fibroblast growth factor-2 (FGF-2). Accordingto some such embodiments, the at least one growth factor is fibroblastgrowth factor-5 (FGF-5). According to some such embodiments, the atleast one growth factor is insulin-like growth factor-1 (IGF-1).According to some such embodiments, the at least one growth factor istransdermal growth factor-beta (TGF-β). According to some suchembodiments, the at least one growth factor is bone morphogenicprotein-2 (BMP-2). According to some such embodiments, the at least onegrowth factor is bone morphogenic protein-7 (BMP-7). According to somesuch embodiments, the at least one growth factor is platelet derivedgrowth factor (PDGF). According to some such embodiments, the at leastone growth factor is vascular endothelial growth factor (VEGF).According to some such embodiments, the at least one growth factor isneural epidermal growth-factor-like 1 (NELL-1).

According to some such embodiments, the at least one growth-inductivecomponent is a demineralized bone matrix (DBM). According to some suchembodiments, the DCB is demineralized autologous bone. According to somesuch embodiments, the DBM is demineralized allogeneic bone. According tosome such embodiments, the DBM is demineralized xenogenic bone.According to some such embodiments, the DBM is derived bydemineralization of cancellous bone. According to some such embodimentsthe DBM is derived by demineralization of cortical bone (i.e.,demineralized cortical bone or DCB). According to some such embodiments,DBM has a residual mineral content of 8% or less (w/w). According tosome such embodiments, DBM has a residual mineral content of 5% or less(w/w). According to some such embodiments, DBM has a residual mineralcontent of 2% or less (w/w). According to some such embodiments, DBM hasa residual mineral content of 1% or less (w/w). According to some suchembodiments, DBM has a residual mineral content of 0.5% or less (w/w).According to some such embodiments, DBM consists essentially ofcollagen, non-collagen proteins such as growth factors, and othernonmineral substances found in the original bone, although notnecessarily in the original quantities.

According to some such embodiments, the at least one growth-inductivecomponent comprises at least one carrier. According to some suchembodiments, the carrier comprises an isotonic solution. According tosome such embodiments, the carrier comprises a sodium chloride solution.According to some such embodiments, the sodium chloride solution is at aconcentration of about 0.1% to about 1%. According to some suchembodiments, the sodium chloride solution is at a concentration of about0.9%. According to some such embodiments, the carrier comprises alactated Ringer's solution. According to some such embodiments, thecarrier comprises PBS. According to some such embodiments, the carriercomprises platelet rich plasma (PRP). According to some suchembodiments, the carrier comprises hyaluronic acid (HA). According tosome such embodiments, the carrier comprises a derivative of HA.According to some such embodiments, the carrier comprises sodiumhyaluronate. Non-limiting examples of HA derivatives include saltderivatives, such as sodium hyaluronate, ester derivatives, such as,ethyl, bezyl, octadecyl ester derivatives. According to some suchembodiments, the carrier comprises thrombin. According to some suchembodiments, the carrier comprises fibrin. According to some suchembodiments, the carrier comprises thrombin and fibrin. According tosome such embodiments, the carrier comprises glycerin. According to somesuch embodiments, the carrier comprises collagen. According to some suchembodiments, the carrier comprises lecithin.

According to some embodiments, the growth-conductive component furthercomprises a growth-inductive component, such as, for example, withoutlimitation, DCB, such that the growth-inductive component representsabout 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about36%, about 37%, about 38%, about 39% or about 40% of the implant.According to some embodiments, at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% of the implant is growth conductive.According to some embodiments, the growth-inductive component of theimplant according to the described invention is at least about 5000 pgof at least one growth-inductive factor, such as, but not limited to, agrowth factor, a cytokine, and a BMP, such as, but not limited to,BMP-2, per gram of a growth-inductive component. The growth-inductivecomponent generally averages about 25000 pg of growth-inductive factorper gram of a growth-inductive component. However, dosage levels arebased on a variety of factors, including the type of injury, the age,weight, sex, medical condition of the patient, the severity of thecondition, the route of administration, and the particular active agentemployed. Thus the dosage regimen may vary widely, but can be determinedroutinely by a physician using standard methods. In prophylactic orpreventative applications of the described invention, pharmaceuticalcompositions or medicaments are administered to a patient susceptibleto, or otherwise at risk of, a disease, disorder or condition in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the onset of the disease, disorder or condition, includingbiochemical, histologic and/or behavioral symptoms of the disease,disorder or condition, its complications, and intermediate pathologicalphenotypes presenting during development of the disease, disorder orcondition.

According to some embodiments, the implant further comprises at leastone cryopreservative. According to some such embodiments, the at leastone cryopreservative is a solution. According to some such embodiments,the cryopreservative is dimethylsulfoxide (DMSO). According to some suchembodiments, the cryopreservative is basal media solution comprisingabout 5% DMSO. According to some such embodiments, the cryopreservativeis basal media solution comprising about 10% DMSO. According to somesuch embodiments, the cryopreservative is basal media solutioncomprising about 15% DMSO. According to some such embodiments, thecryopreservative is fetal bovine serum comprising about 5% DMSO.According to some such embodiments, the cryopreservative is fetal bovineserum comprising about 10% DMSO. According to some such embodiments, thecryopreservative is a human serum comprising about 15% DMSO. Accordingto some such embodiments, the cryopreservative is human serum comprisingabout 5% DMSO. According to some such embodiments, the cryopreservativeis human serum comprising about 10% DMSO. According to some suchembodiments, the cryopreservative is ethylene glycol. According to somesuch embodiments, the cryopreservative is propylene glycol. According tosome such embodiments, the cryopreservative is glycerol.

Orthopedic Implant

According to another embodiment, the described invention provides anorthopedic implant comprising

a plurality of particles comprising at least one growth-conductivematrix or at least one fragment thereof; and

a viable population of tissuegenic cells adherent to and resident in thegrowth-conductive matrix.

According to one embodiment, the plurality of particles or piecescomprising at least one growth-conductive matrix can be of any form.According to some embodiments, the plurality of particles or piecescomprises a plurality of sheets. According to some embodiments, theplurality of particles or pieces comprises a slurry form. According tosome embodiments, the plurality of particles or pieces comprises a pasteform. According to some embodiments, the plurality of particles orpieces comprises a three-dimensional form. According to someembodiments, the three-dimensional form is selected from the groupconsisting of a block, a dowel, a sheet, and a combination thereof.According to some such embodiments, the three-dimensional form comprisesa block. According to some such embodiments, the three-dimensional formcomprises a dowel. According to some such embodiments, thethree-dimensional form comprises a sheet.

According to another embodiment, the implant is an implantablecomposition comprising a growth-conductive matrix (e.g., anosteoconductive matrix including bone), a viable population oftissuegenic cells (e.g., osteogenic cells), and a growth-inductivematrix (e.g., demineralized bone matrix). In an embodiment, a secondgrowth-inductive matrix (e.g., a second demineralized bone matrix) isprovided separately from the aforesaid implantable composition foraddition to the implantable composition at a later time.

2. Method of Fabricating an Implant Using a Tissue-Derived MatrixContaining Endogenous Tissuegenic Cells

According to another aspect, the described invention provides a methodof fabricating an implant, the method comprising steps:

(a) providing at least one growth-conductive matrix wherein thegrowth-conductive matrix comprises at least one viable population oftissuegenic cells endogenous to the tissue, wherein the tissuegeniccells are adherent to and resident in an endogenous milieu of thegrowth-conductive matrix;

(b) separating the at least one growth-conductive matrix of (a) togenerate a plurality of separated matrix pieces comprising the at leastone viable population of tissuegenic cells adherent to and resident inthe endogenous milieu of the growth-conductive matrix, wherein arelative frequency of a stem cell subtype in the at least one viabletissuegenic cell population of step (b) is of a relative frequencysubstantially similar to that found in the growth-conductive matrix ofstep (a);

(c) rinsing the plurality of separated matrix pieces of (b) comprisingthe at least one viable population of tissuegenic cells adherent to andresident in the endogenous milieu of the growth-conductive matrix of (b)to form a plurality of rinsed separated matrix pieces comprising the atleast one viable population of tissuegenic cells adherent to andresident in the the endogenous milieu of the growth-conductive matrix of(b), wherein a relative frequency of a stem cell subtype in the at leastone viable tissuegenic cell population of step (c) is of a relativefrequency substantially similar to that found in the growth-conductivematrix of step (a);

(d) collecting the plurality of rinsed separated matrix pieces of (c)comprising the at least one viable population of tissuegenic cellsadherent to and resident in the endogenous milieu of thegrowth-conductive matrix of (c) wherein the at least one viabletissuegenic cell population is of a relative frequency substantiallysimilar to that found in the growth-conductive matrix of step (a);

(e) packaging the plurality of collected rinsed separated matrix piecesof (d) comprising at least one viable population of tissuegenic cellsadherent to and resident in the endogenous milieu of thegrowth-conductive matrix of (c) wherein the at least one viabletissuegenic cell population is of a relative frequency substantiallysimilar to that found in the growth-conductive matrix of step (a) toform the implant.

Providing Step (a): Providing at Least One Growth-Conductive MatrixWherein the Growth-Conductive Matrix Comprises at Least One ViablePopulation of Tissuegenic Cells Endogenous to the Tissue, Wherein theTissuegenic Cells are Adherent to and Resident in an Endogenous Milieuof the Growth-Conductive Matrix

According to some embodiments, providing step (a) comprises excising thetissue from its source. According to some embodiments, providing step(a) comprises removing the tissue from its source. According to someembodiments, providing step (a) comprises isolating the tissue from itssource. According to some embodiments, providing step (a) comprisesrecovering the tissue from its source.

According to some embodiments, providing step (a) is at a temperature ofabout 25° C. According to some embodiments, providing step (a) is at atemperature of about 4° C. to about 10° C. According to someembodiments, providing step (a) is at an ambient temperature.

According to some embodiments, the tissue is rinsed with a liquid priorto being separated into pieces to reduce bioburden levels on the surfaceof the tissue. According to some embodiments, the liquid comprisesphosphate buffered saline (PBS). According to some embodiments, theliquid comprises acetic acid. According to some embodiments, the liquidcomprises peracetic acid.

According to another embodiment, the method of fabricating an implant,further comprises step (f) supplementing the growth-conductive matrix ofstep (a) with at least one growth-inductivecomponent. According to somesuch embodiments, the at least one growth-inductive component comprisesat least one growth-inductive factor. According to some suchembodiments, the at least one growth-inductive factor comprises at leastone growth factor. According to some such embodiments, the at least onegrowth factor is fibroblast growth factor-2 (FGF-2). According to somesuch embodiments, the at least one growth factor is fibroblast growthfactor-5 (FGF-5). According to some such embodiments, the at least onegrowth factor is insulin-like growth factor 1 (IGF-1). According to somesuch embodiments, the at least one growth factor is transforming growthfactor beta (TGF-β). According to some such embodiments, the at leastone growth factor is bone morphogenic protein-2 (BMP-2). According tosome such embodiments, the at least one growth factor is bonemorphogenic protein-7 (BMP-7). According to some such embodiments, theat least one growth factor is platelet-derived growth factor (PDGF).According to some such embodiments, the at least one growth factor isvascular endothelial growth factor (VEGF). According to some suchembodiments, the at least one growth factor is neural epidermalgrowth-factor-like 1 (NELL-1). According to another embodiment, therinsed osteoconductive matrix particles are supplemented with at leastone cytokine.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to oneembodiment, the at least one growth-inductive component is endogenous tothe at least one growth-conductive matrix. According to one embodiment,the tissuegenic cells adherent to and resident in the endogenous milieuof the growth conductive matrix secrete the at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductive component is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells. According to one embodiment, the at leastone growth-inductive component comprises demineralized cortical bone.

Separating Step (b): Separating the at Least One Growth-ConductiveMatrix of (a) to Generate a Plurality of Separated Matrix PiecesComprising the at Least One Viable Population of Tissuegenic CellsAdherent to and Resident in the Endogenous Milieu of theGrowth-Conductive Matrix, Wherein a Relative Frequency of a Stem CellSubtype in the at Least One Viable Tissuegenic Cell Population of Step(b) is of a Relative Frequency Substantially Similar to that Found inthe Growth-Conductive Matrix of Step (a)

According to some embodiments, separating step (b) comprises mincing thetissue. According to some embodiments, separating step (b) comprisescutting the tissue. According to some embodiments, separating step (b)comprises a slicing step. According to some embodiments, separating step(b) comprises milling the tissue. According to some embodiments,separating step (b) comprises homogenizing the tissue.

According to some embodiments, separating step (b) is at a temperatureof about 25° C. According to some embodiments, separating step (b) is ata temperature of about 4° C. to about 10° C. According to someembodiments, separating step (b) is at an ambient temperature.

According to some embodiments, the plurality of separatedgrowth-conductive matrix pieces of step (b) comprises a plurality ofmatrix particles.

According to some embodiments, the plurality of growth-conductive matrixpieces can be of virtually any shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a similar shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of a circular shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a square shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of a polygonal shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a rectangular shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of a triangular shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a octagonal shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of irregular shapes. According to some embodiments, theplurality of growth-conductive pieces are of an elongated shape (e.g.,as a fiber). According to some embodiments, the plurality ofgrowth-conductive matrix pieces are of an amorphous shape.

According to some embodiments, the plurality of growth-conductive matrixpieces comprises at least one growth-conductive matrix piece whoselongest dimension (which is used herein as an equivalent term to“maximum dimension”) is of about 10 μm to about 20 cm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 10μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 20 μm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 30 μm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about40 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 50 μm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 100 μm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about150 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 200 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 250μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 300 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 350μm. According to some such embodiments, the at least growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 400 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 450 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 500μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 550 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 600μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 650 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 700μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 750 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 800μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 850 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 900μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 950 μm. According to some embodiments, theat least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is in the range of about 100 μm toabout 1000 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 1 mm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 2 mm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 3 mm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 4 mm. According to some such embodiments, the at least oneosteocogrowth-conductive matrix piece is a growth-conductive matrixpiece whose longest dimension is of about 5 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 6 mm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 7 mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 8 mm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 9 mm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 10mm. According to some such embodiments the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is in the range of about 1 mm to about 10 mm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 50 mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 100 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 200mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a osteocongrowth-conductive matrixpiece whose longest dimension is of about 300 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 400mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 500 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 600mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 700 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 800mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 900 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 1 cm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 2 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 3 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 4 cm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 5 cm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 6 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 7 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 8 cm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 9 cm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 10 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 11 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 12 cm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about13 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 14 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 15 cm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about16 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 17 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 18 cm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about19 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 20 cm.

According to some embodiments, the plurality of separatedgrowth-conductive matrix pieces can be of any form. According to someembodiments, the plurality of separated growth-conductive matrix piecescomprises a plurality of sheets. According to some embodiments, theplurality of separated growth-conductive matrix pieces comprises apowder form. According to some embodiments, the plurality of separatedgrowth-conductive matrix pieces comprises a slurry form. According tosome embodiments, the plurality of separated growth-conductive matrixpieces comprises a three-dimensional form. According to someembodiments, the three-dimensional form is selected from the groupconsisting of a block, a dowel, a sheet, and a combination thereof.According to some such embodiments, the three-dimensional form comprisesa block. According to some such embodiments, the three-dimensional formcomprises a dowel. According to some such embodiments, thethree-dimensional form comprises a sheet.

According to another embodiment, the method of fabricating an implantfurther comprises step (f) supplementing the plurality of separatedgrowth-conductive matrix pieces of step (b) with at least onegrowth-inductive component. According to some such embodiments, the atleast one growth-inductive component comprises at least onegrowth-inductive factor. According to some such embodiments, the atleast one growth-inductive factor comprises at least one growth factor.According to some such embodiments, the at least one growth factor isfibroblast growth factor-2 (FGF-2). According to some such embodiments,the at least one growth factor is fibroblast growth factor-5 (FGF-5).According to some such embodiments, the at least one growth factor isinsulin-like growth factor 1 (IGF-1). According to some suchembodiments, the at least one growth factor is transforming growthfactor beta (TGF-β). According to some such embodiments, the at leastone growth factor is bone morphogenic protein-2 (BMP-2). According tosome such embodiments, the at least one growth factor is bonemorphogenic protein-7 (BMP-7). According to some such embodiments, theat least one growth factor is platelet-derived growth factor (PDGF).According to some such embodiments, the at least one growth factor isvascular endothelial growth factor (VEGF). According to some suchembodiments, the at least one growth factor is neural epidermalgrowth-factor-like 1 (NELL-1). According to another embodiment, therinsed growth conductive matrix particles are supplemented with at leastone cytokine.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to oneembodiment, the at least one growth-inductive component is endogenous tothe at least one growth-conductive matrix. According to one embodiment,the tissuegenic cells adherent to and resident in the endogenous milieuof the growth conductive matrix secrete the at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductive component is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells.

According to one embodiment, the at least one growth-inductive componentcomprises demineralized bone matrix, which may be demineralized corticalbone. According to some embodiments, the demineralized bone matrix isprovided as a plurality of pieces of demineralized bone matrix derivedfrom a single piece of demineralized bone matrix. According to someembodiments, the demineralized bone matrix is provided as a plurality ofpieces of demineralized bone matrix derived from a plurality of piecesof bone tissue.

According to some embodiments, the pieces of bone tissue that are to bedemineralized have a longest dimension (which is used herein as anequivalent term to “maximum dimension”) that is measurable prior todemineralization of the pieces of bone. All such longest dimensionsrelated in this paragraph are the longest dimension of a piece of bonetissue prior to demineralization. According to some embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 10 μm to about 20 cm.According to some such embodiments, the plurality of pieces of bonetissue includes a piece of bone tissue whose longest dimension is ofabout 10 μm. According to some such embodiments, the plurality of piecesof bone tissue includes a piece of bone tissue whose longest dimensionis of about 20 μm. According to some such embodiments, the plurality ofpieces of bone tissue includes at least one piece of bone tissue whoselongest dimension is of about 30 μm. According to some such embodiments,the plurality of pieces of bone tissue includes at least one piece ofbone tissue whose longest dimension is of about 40 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 50μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 100 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 150 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 200μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 250 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 300 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 350μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 400 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 450 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 500μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 550 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 600 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 650μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 700 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 750 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 800μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 850 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 900 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 950μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is in the range of about 100 μm to about 1000 μm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose longest dimension is of about 1mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 2 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 3 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 4 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 5 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 6 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 7 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 8 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 9 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 10 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is in the range of about 1 mm to about 10 mm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose longest dimension is of about 50mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 100 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 200 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 300mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 400 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 500 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 600mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 700 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 800 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 900mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 1 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 2 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 3 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 4 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 5 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 6 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 7 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 8 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 9 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 10 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 11 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 12 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 13 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 14 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 15 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 16 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 17 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 18 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 19 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 20 cm.

According to some embodiments, the pieces of bone tissue that are to bedemineralized have a shortest dimension (which is used herein as anequivalent term to “minimum dimension”) that is measurable prior todemineralization of the pieces of bone. All such shortest dimensionsrelated in this paragraph are the shortest dimension of a piece of bonetissue prior to demineralization. According to some embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 10 μm to about 20 cm.According to some such embodiments, the plurality of pieces of bonetissue includes a piece of bone tissue whose shortest dimension is ofabout 10 μm. According to some such embodiments, the plurality of piecesof bone tissue includes a piece of bone tissue whose shortest dimensionis of about 20 μm. According to some such embodiments, the plurality ofpieces of bone tissue includes at least one piece of bone tissue whoseshortest dimension is of about 30 μm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 40 μm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 50 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 100 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 150μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 200 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 250 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 300μm. According to some embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue having a shortestdimension in the range of about 5 μm to about 300 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 350μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 400 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 450 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 500μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 550 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 600 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 650μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 700 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 750 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 800μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 850 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 900 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 950μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is in the range of about 100 μm to about 1000 μm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose shortest dimension is of about 1mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 2 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 3 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 4 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 5 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 6 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 7 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 8 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 9 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 10 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is in the range of about 1 mm to about 10 mm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose shortest dimension is of about50 mm. According to some such embodiments, the plurality of pieces ofbone tissue includes at least one piece of bone tissue whose shortestdimension is of about 100 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 200 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 300mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 400 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 500 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 600mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 700 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 800 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 900mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 1 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 2 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 3 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 4 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 5 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 6 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 7 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 8 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 9 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 10 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 11 cm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 12cm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 13 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 14 cm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 15cm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 16 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 17 cm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 18cm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 19 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 20 cm.

Rinsing Step (c): Rinsing the Plurality of Separated Matrix Pieces of(b) Comprising the at Least One Viable Population of Tissuegenic CellsAdherent to and Resident in the Endogenous Milieu of theGrowth-Conductive Matrix of (b) to Form a Plurality of Rinsed SeparatedMatrix Pieces Comprising the at Least One Viable Population ofTissuegenic Cells Adherent to and Resident in the the Endogenous Milieuof the Growth-Conductive Matrix of (b), Wherein a Relative Frequency ofa Stem Cell Subtype in the at Least One Viable Tissuegenic CellPopulation of Step (c) is of a Relative Frequency Substantially Similarto that Found in the Growth-Conductive Matrix of Step (a)

According to another embodiment, rinsing step (c) comprises admixing theplurality of separated growth-conductive matrix pieces of (b) comprisingthe at least one viable population of tissuegenic cells adherent to andresident in the endogenous milieu of the growth-conductive matrix with aliquid, wherein the liquid is a buffer. According to some embodiments,the liquid comprises a physiological saline solution. According to someembodiments, the liquid comprises a buffered isotonic solution.According to some embodiments, the physiological saline solution is aphosphate buffered saline (PBS) solution. According to some embodiments,the liquid comprises an acetic acid solution. According to someembodiments, the liquid comprises an ammonium chloride solution.According to some embodiments, the ammonium chloride solution furthercomprises ethylenediaminetetraacetic acid (EDTA). According to some suchembodiments, the EDTA is at a concentration from about 0.1 mM to about0.5 mM.

According to some embodiments, rinsing step (c) comprises admixing theplurality of separated growth-conductive matrix pieces of (b) with aliquid at a temperature of about 4° C. to about 10° C. According to someembodiments, rinsing step (c) comprises admixing the plurality ofseparated growth-conductive matrix pieces of (b) with a liquid at atemperature of about 25° C. According to some embodiments, rinsing step(c) comprises admixing the plurality of separated growth-conductivematrix pieces of (b) with a liquid at an ambient temperature.

According to one embodiment, rinsing step (c) comprises admixing theplurality of separated growth-conductive matrix pieces of (b) with aliquid such that unwanted cells are removed. According to oneembodiment, the plurality of growth-conductive matrix pieces comprises aplurality of growth conductive matrix pieces from which unwanted cellshave been removed. According to some embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth conductive matrix is immune privileged.The term “immune privileged” as used herein refers to the characteristicof tissuegenic cells by which there is no induction of an immuneresponse upon transplantation of such cells.

According to another embodiment, the method of fabricating an implantfurther comprises step (f) supplementing the plurality of rinsedgrowth-conductive matrix pieces of step (c) with at least onegrowth-inductivecomponent. According to some such embodiments, the atleast one growth-inductivecomponent comprises at least onegrowth-inductivefactor. According to some such embodiments, the at leastone growth-inductivefactor comprises at least one growth factor.According to some such embodiments, the at least one growth factor isfibroblast growth factor-2 (FGF-2). According to some such embodiments,the at least one growth factor is fibroblast growth factor-5 (FGF-5).According to some such embodiments, the at least one growth factor isinsulin-like growth factor 1 (IGF-1). According to some suchembodiments, the at least one growth factor is transforming growthfactor beta (TGF-β). According to some such embodiments, the at leastone growth factor is bone morphogenic protein-2 (BMP-2). According tosome such embodiments, the at least one growth factor is bonemorphogenic protein-7 (BMP-7). According to some such embodiments, theat least one growth factor is platelet-derived growth factor (PDGF).According to some such embodiments, the at least one growth factor isvascular endothelial growth factor (VEGF). According to some suchembodiments, the at least one growth factor is neural epidermalgrowth-factor-like 1 (NELL-1). According to another embodiment, therinsed growth conductive matrix pieces are supplemented with at leastone cytokine.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to oneembodiment, the at least one growth-inductive component is endogenous tothe at least one growth-conductive matrix. According to one embodiment,the tissuegenic cells adherent to and resident in the endogenous milieuof the growth conductive matrix secrete the at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductivecomponent is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells. According to one embodiment, the at leastone growth-inductive component comprises demineralized bone matrix,which may be demineralized cortical bone.

Packaging Step (e): packaging the plurality of collected rinsedseparated matrix pieces of (d) comprising at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix of (c) wherein the at least one viabletissuegenic cell population is of a similar relative frequency as foundin the growth-conductive matrix of step (a) to form the implant.

Cryopreservation is used for the long-term preservation of varioustissues and cells. According to some embodiments, tissuegenic cellsderived from a tissue can be cryopreserved, reconstituted, and seededonto an isolated matrix. According to some embodiments, tissuegeniccells derived from a tissue can be cryopreserved, reconstituted, andseeded onto an isolated matrix to promote tissue genesis in vitro and invivo. According to some such embodiments, the isolated matrix is ademineralized bone matrix, which may be demineralized cortical bone.

Water is the major component of all living cells and must be availablefor the chemical processes of life to occur; cellular metabolism stopswhen all water in the system is converted to ice. For reconstitution,most cells are warmed quickly until complete thawing is achieved (e.g.,a 37° C. water bath) in order to prevent recrystallization of ice.

The detrimental effects of ice crystal formation and increased soluteconcentration can be reduced by using cryoprotective additives orchemicals that protect cells during freezing. Commonly usedcryoprotective agents include, but are not limited to, dimethylsulfoxide(DMSO), ethylene glycol, propylene glycol, 2-Methyl-2.4-pentanediol(MPD), sucrose, and glycerol. Examples of cryopreservation solutionsthat can be used in preserving a tissue or a matrix include, but are notlimited to, a commercially available basal media solution such as,Mesencult (Stem Cell Technologies), or Hyclone AdvanceStem, Fetal BovineSerum with 5-15% DMSO, Bovine Serum Albumin with 5-15% DMSO, Human SerumAlbumin with 5-15% DMSO, Aedesta (Cell Preservation Solutions),LiforCell (Lifeblood Medical), ethylene glycol, propylene glycol, andglycerol.

According to one embodiment, the growth-conductive matrix of step (a),step (b), step (c) or step (d) comprises a frozen growth-conductivematrix. According to one embodiment, the growth-conductive matrix ofstep (a), step (b), step (c) or step (d) comprises a freshgrowth-conductive matrix.

According to some embodiments, packaging step (e) comprises admixing theplurality of collected rinsed separated matrix pieces of (d) with atleast one cryopreservative. According to some such embodiments,packaging step (e) comprises admixing the plurality of collected rinsedseparated matrix pieces of (d) with at least one cryopreservative,wherein the cryopreservative is a solution. According to some suchembodiments, packaging step (e) comprises admixing the plurality ofcollected rinsed separated matrix pieces of (d) with at least onecryopreservative, wherein the cryopreservative is dimethylsulfoxide(DMSO). According to some such embodiments, packaging step (e) comprisesadmixing the plurality of collected rinsed separated matrix pieces of(d) with at least one cryopreservative, wherein the cryopreservative isa basal media solution comprising about 5% DMSO. According to some suchembodiments, packaging step (e) comprises admixing the plurality ofcollected rinsed separated matrix pieces of (d) with at least onecryopreservative, wherein the cryopreservative is a basal media solutioncomprising about 10% DMSO. According to some such embodiments, packagingstep (e) comprises admixing the plurality of collected rinsed separatedmatrix pieces of (d) with at least one cryopreservative, wherein thecryopreservative is a basal media solution comprising about 15% DMSO.According to some such embodiments, packaging step (e) comprisesadmixing the plurality of collected rinsed separated matrix pieces of(d) with at least one cryopreservative, wherein the cryopreservativecomprises fetal bovine serum comprising about 5% DMSO. According to somesuch embodiments, packaging step (e) comprises admixing the plurality ofcollected rinsed separated matrix pieces of (d) with at least onecryopreservative, wherein the cryopreservative comprises fetal bovineserum comprising about 10% DMSO. According to some such embodiments,packaging step (e) comprises admixing the plurality of collected rinsedseparated matrix pieces of (d) with at least one cryopreservative,wherein the cryopreservative comprises fetal bovine serum comprisingabout 15% DMSO. According to some such embodiments, packaging step (e)comprises admixing the plurality of collected rinsed separated matrixpieces of (d) with at least one cryopreservative, wherein thecryopreservative comprises bovine serum albumin (BSA) comprising about5% DMSO. According to some such embodiments, packaging step (e)comprises admixing the plurality of collected rinsed separated matrixpieces of (d) with at least one cryopreservative, wherein thecryopreservative comprises BSA comprising about 10% DMSO. According tosome such embodiments, packaging step (e) comprises admixing theplurality of collected rinsed separated matrix pieces of (d) with atleast one cryopreservative, wherein the cryopreservative comprises BSAcomprising about 15% DMSO. According to some such embodiments, packagingstep (e) comprises admixing the plurality of collected rinsed separatedmatrix pieces of (d) with at least one cryopreservative, wherein thecryopreservative comprises human serum comprising about 5% DMSO.According to some such embodiments, packaging step (e) comprisesadmixing the plurality of collected rinsed separated matrix pieces of(d) with at least one cryopreservative, wherein the cryopreservativecomprises human serum comprising about 10% DMSO. According to some suchembodiments, packaging step (e) comprises admixing the plurality ofcollected rinsed separated matrix pieces of (d) with at least onecryopreservative, wherein the cryopreservative comprises human serumcomprising about 15% DMSO. According to some such embodiments, packagingstep (e) comprises admixing the plurality of collected rinsed separatedmatrix pieces of (d) with at least one cryopreservative, wherein thecryopreservative comprises human serum albumin comprising about 5% DMSO.According to some such embodiments, packaging step (e) comprisesadmixing the plurality of collected rinsed separated matrix pieces of(d) with at least one cryopreservative, wherein the cryopreservativecomprises human serum albumin comprising about 10% DMSO. According tosome such embodiments, packaging step (e) comprises admixing theplurality of collected rinsed separated matrix pieces of (d) with atleast one cryopreservative, wherein the cryopreservative comprises humanserum albumin comprising about 15% DMSO. According to some suchembodiments, packaging step (e) comprises admixing the plurality ofcollected rinsed separated matrix pieces of (d) with at least onecryopreservative, wherein the cryopreservative comprises ethyleneglycol. According to some such embodiments, packaging step (e) comprisesadmixing the plurality of collected rinsed separated matrix pieces of(d) with at least one cryopreservative, wherein the cryopreservativecomprises propylene glycol. According to some such embodiments,packaging step (e) comprises admixing the plurality of collected rinsedseparated matrix pieces of (d) with at least one cryopreservative,wherein the cryopreservative comprises glycerol.

According to some embodiments of the described invention, the packagedimplant can be preserved for an extended period of time by slowlycooling the packaged implant in the presence of a cryoprotective agentand by storing at ultra low temperatures. According to some suchembodiments, packaging step (e) comprises freezing the the plurality ofgrowth-conductive matrix pieces to at least a temperature of −80° C.According to some such embodiments, packaging step (e) comprisesfreezing the plurality of collected growth-conductive matrix pieces at acontrolled freezing rate. According to some such embodiments, thecontrolled freezing rate is a controlled freezing rate of about 0.5° C.per minute to about 10° C. per minute. According to some suchembodiments, the controlled freezing rate is a controlled freezing rateof about 1° C. per minute until about −100° C.

According to some such embodiments, the method of fabricating an implantfurther comprises (f) thawing the plurality of rinsed growth-conductivematrix pieces of step (e) to form a plurality of thawed rinsedgrowth-conductive matrix pieces. According to some such embodiments, theplurality of growth-conductive matrix pieces comprises the implant.

According to another embodiment, the packaged implant comprising theplurality of packaged growth-conductive matrix pieces of (e) comprisesat least one viable population of tissuegenic cells adherent to andresident in the endogenous milieu of the growth-conductive matrix.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from an adipose tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from an amnion tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from an artery tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a bone tissue. Accordingto some such embodiments, the at least one viable population oftissuegenic cells adherent to and resident in the endogenous milieu ofthe growth-conductive matrix is derived from a cartilage tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a chorion tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a colon tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a dental tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a dermal tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a duodenal tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from an epithelial tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a fascial tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a gastrointestinaltissue. According to some such embodiments, the at least one viablepopulation of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from agrowth plate tissue. According to some such embodiments, the at leastone viable population of tissuegenic cells adherent to and resident inthe endogenous milieu of the growth-conductive matrix is derived from anintervertebral disc tissue. According to some such embodiments, the atleast one viable population of tissuegenic cells adherent to andresident in the endogenous milieu of the growth-conductive matrix isderived from an intestinal mucosal tissue. According to some suchembodiments, the at least one viable population of tissuegenic cellsadherent to and resident in the endogenous milieu of thegrowth-conductive matrix is derived from an intestinal serosal tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a kidney tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a ligament tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a liver tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a lung tissue. Accordingto some such embodiments, the at least one viable population oftissuegenic cells adherent to and resident in the endogenous milieu ofthe growth-conductive matrix is derived from a mammary tissue. Accordingto some such embodiments, the at least one viable population oftissuegenic cells adherent to and resident in the endogenous milieu ofthe growth-conductive matrix is derived from a meniscal tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a muscle tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a nerve tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from an ovarian tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a pancreatic tissue.According to some such embodiments, the at least one viable populationof tissuegenic cells adherent to and resident in the endogenous milieuof the growth-conductive matrix is derived from a parenchymal organtissue. According to some such embodiments, the at least one viablepopulation of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from apericardial tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from aperiosteal tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from aperitoneal tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from aplacental tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from a skintissue. According to some such embodiments, the at least one viablepopulation of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from aspleen tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from astomach tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from atendon tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from asynovial tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from atendon tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from atestes tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from anumbilical cord tissue. According to some such embodiments, the at leastone viable population of tissuegenic cells adherent to and resident inthe endogenous milieu of the growth-conductive matrix is derived from aurological tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from avascular tissue. According to some such embodiments, the at least oneviable population of tissuegenic cells adherent to and resident in theendogenous milieu of the growth-conductive matrix is derived from a veintissue.

According to another embodiment, the method of fabricating an implantfurther comprises step (f) supplementing the plurality of rinsedgrowth-conductive matrix pieces of step (c) with at least onegrowth-inductive component. According to some such embodiments, the atleast one growth-inductive component comprises at least onegrowth-inductive factor. According to some such embodiments, the atleast one growth-inductive factor comprises at least one growth factor.According to some such embodiments, the at least one growth-inductivecomponent comprises a demineralized cortical bone. According to somesuch embodiments, the at least one growth factor is fibroblast growthfactor-2 (FGF-2). According to some such embodiments, the at least onegrowth factor is fibroblast growth factor-5 (FGF-5). According to somesuch embodiments, the at least one growth factor is insulin-like growthfactor 1 (IGF-1). According to some such embodiments, the at least onegrowth factor is transforming growth factor beta (TGF-β). According tosome such embodiments, the at least one growth factor is bonemorphogenic protein-2 (BMP-2). According to some such embodiments, theat least one growth factor is bone morphogenic protein-7 (BMP-7).According to some such embodiments, the at least one growth factor isplatelet-derived growth factor (PDGF). According to some suchembodiments, the at least one growth factor is vascular endothelialgrowth factor (VEGF). According to some such embodiments, the at leastone growth factor is neural epidermal growth-factor-like 1 (NELL-1).According to another embodiment, the rinsed osteoconductive matrixparticles are supplemented with at least one cytokine.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductivecomponent other than cells. According to one embodiment,the at least one growth-inductive component is endogenous to the atleast one growth-conductive matrix. According to one embodiment, thetissuegenic cells adherent to and resident in the endogenous milieu ofthe growth conductive matrix secrete the at least one growth-inductivecomponent. According to one embodiment, the at least onegrowth-inductive component is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells.

3. Method of Fabricating an Implant Using a Tissue-Derived MatrixContaining Reseeded Tissuegenic Cells Caused to be in Contact with theMatrix

According to another aspect, the described invention provides a methodof fabricating an implant, the method comprising steps:

(i) providing at least one first growth-conductive matrix or a fragmentthereof derived from human tissue, wherein the growth-conductive matrixcomprises at least one viable population of tissuegenic cells endogenousto the tissue, wherein the tissuegenic cells are adherent to andresident in the endogenous milieu of the first growth-conductive matrix;

(ii) isolating from the at least one growth-conductive matrix of (i) aplurality of the at least one viable isolated population of tissuegeniccells;

(iii) processing a second growth-conductive matrix comprising adecellularized growth-conductive matrix to generate a plurality ofpieces;

(iv) seeding the decellularized growth-conductive matrix pieces of (iii)with the plurality of at least one viable isolated population oftissuegenic cells of (ii) to form a plurality of reseededgrowth-conductive matrix pieces, wherein the at least one viablepopulation of tissuegenic cells is caused to be in contact with thegrowth-conductive matrix;

(v) collecting the plurality of reseeded growth-conductive matrix piecesof (iv) comprising the at least one viable population of tissuegeniccells caused to be in contact with the growth-conductive matrix of (iv),wherein the at least one viable tissuegenic cell population is of arelative frequency substantially similar to that found in thegrowth-conductive matrix of step (i);

(vi) packaging the plurality of collected reseeded growth-conductivematrix pieces of (v) comprising the at least one viable population oftissuegenic cells caused to be in contact with the growth-conductivematrix of (iv), wherein the at least one viable tissuegenic cellpopulation is of a relative frequency substantially similar to thatfound in the growth-conductive matrix of step (i) to form an implant.

Isolating Step (ii): Isolating from the at Least One Growth-ConductiveMatrix of (i) a Plurality of the at Least One Viable Isolated Populationof Tissuegenic Cells

According to some embodiments, isolating step (ii) comprises isolatingfrom the at least one growth-conductive matrix of (i) a plurality of theat least one viable isolated population of tissuegenic cells.

According to some such embodiments, isolating step (ii) comprises thesteps: (1) washing of the tissue of (i); (2) optionally digesting thewashed tissue of (1); (3) fractionating the tissue of (1) or thedigested tissue of (2) into an isolated fraction comprising at least oneviable population of tissuegenic cells; (4) washing the isolatedfraction comprising at least one viable population of tissuegenic cells;and (5) filtering the washed fraction comprising at least one viablepopulation of tissuegenic cells to generate a plurality of the at leastone viable isolated population of tissuegenic cells.

According to some embodiments, isolating step (ii) is at a temperatureof about 25° C. According to some embodiments, isolating step (ii) is ata temperature of about 4° C. to about 10° C. According to someembodiments, isolating step (ii) is at an ambient temperature. Accordingto some embodiments, washing step (1) is at a temperature of about 25°C. According to some embodiments, washing step (1) is at a temperatureof about 4° C. to about 10° C. According to some embodiments, washingstep (1) is at an ambient temperature. According to some embodiments,digesting step (2) is at a temperature of about 25° C. According to someembodiments, digesting step (2) is at a temperature of about 4° C. toabout 10° C. According to some embodiments, digesting step (2) is at anambient temperature. According to some embodiments, fractionating step(3) is at a temperature of about 25° C. According to some embodiments,fractionating step (3) is at a temperature of about 4° C. to about 10°C. According to some embodiments, fractionating step (3) is at anambient temperature. According to some embodiments, washing step (4) isat a temperature of about 25° C. According to some embodiments, washingstep (4) is at a temperature of about 4° C. to about 10° C. According tosome embodiments, washing step (4) is at an ambient temperature.According to some embodiments, filtering step (5) is at a temperature ofabout 25° C. According to some embodiments, filtering step (5) is at atemperature of about 4° C. to about 10° C. According to someembodiments, filtering step (5) is at an ambient temperature.

According to some embodiments, washing step (1) comprise washing of thetissue of (i) with a liquid. According to some embodiments, washing step(1) comprises washing of the tissue of (i) with a liquid wherein theliquid is a buffer. According to some embodiments, washing step (1)comprises washing of the tissue of (i) with a buffer, wherein the bufferis a physiological saline solution. According to some embodiments,washing step (1) comprises washing of the tissue of (i) with a buffer,wherein the buffer is a buffered isotonic solution. According to someembodiments, washing step (1) comprises washing of the tissue of (i)with a buffer, wherein the buffer is a buffered salt solution. Accordingto some embodiments, the buffered salt solution is a Hank's bufferedsalt solution (HBSS). According to some embodiments, the buffered saltsolution is a phosphate buffered saline (PBS) solution.

According to some embodiments, optional digesting step (2) comprisesdigesting the washed tissue of (1) with an enzyme to yield a crudeextract. According to some embodiments, digesting step (2) comprisesdigesting the washed tissue of (1) with an enzyme, wherein the enzyme iscollagenase. According to some embodiments, digesting step (2) comprisesdigesting the washed tissue of (1) with an enzyme, wherein the enzyme istrypsin. According to some such embodiments, isolating step (ii)optionally comprises a step comprising admixing the crude extract ofstep (2) with a neutralizing solution. According to some suchembodiments, neutralizing solution is a basal media solution. Accordingto some embodiments, the neutralizing solution comprises serum.According to some embodiments, the serum comprises a human serum.According to some such embodiments, fractionating step (3) comprisesisolating from the optionally neutralized crude extract of step (2) toyield a fraction comprising at least one viable population oftissuegenic cells. According to some such embodiments, fractionatingstep (3) comprises a centrifugation step. According to some suchembodiments, centrifugation step generates a pellet fraction comprisingat least one viable population of tissuegenic cells. According to somesuch embodiments, fractionating step (3) further comprises resuspendingthe pellet in a liquid. According to some embodiments, the liquid is abasal media solution. According to some embodiments, the resuspendedpellet fraction comprising at least one viable population of tissuegeniccells is further digested with an enzyme to yield a double-digestedextract comprising at least one viable population of tissuegenic cells.According to some embodiments, the double-digested extract comprisingthe at least one viable population of tissuegenic cells is fractionatedto yield an isolated fraction comprising the at least one viablepopulation of tissuegenic cells.

According to some embodiments, fractionating step (3) comprisesisolating a fraction comprising the at least one viable population oftissuegenic cells from the tissue of step (1), using an immunoseparationstep. The immunoseparation step can be any cell based immunoseparationmethods well known in the art, including but not limited toimmunoprecipitation using magnetic beads, affinity chromatography,fluorescence activated cell sorting (FACS) or flow cytometry.

According to some embodiments, the isolated fraction comprising the atleast one viable population of tissuegenic cells comprises a nonexpandedpopulation of tissuegenic cells. According to some embodiments, theisolated fraction comprising the at least one viable population oftissuegenic cells is further expanded to generate an expanded populationof tissuegenic cells. Any method of clonal expansion well known in theart can be used.

According to some embodiments, isolating step (ii) further comprises awashing step, wherein the isolated fraction of the at least one viablepopulation of tissuegenic cells is further washed with a liquid.According to some embodiments, the liquid comprises a buffered isotonicsolution. According to some embodiments, the liquid comprises a bufferedsalt solution. According to some embodiments, the buffered salt solutioncomprises a PBS solution. According to some embodiments, isolating step(ii) further comprises a filtering step, wherein the isolated fractionof the at least one viable population of tissuegenic cells is filtered.

According to another embodiment, the method of fabricating an implantfurther comprises step (vii) supplementing the plurality of the at leastone viable isolated population of tissuegenic cells. According to somesuch embodiments, the at least one growth-inductive component comprisesat least one growth-inductive factor. According to some suchembodiments, the at least one growth-inductive factor comprises at leastone growth factor. According to some such embodiments, the at least onegrowth-inductive component comprises a demineralized bone matrix, whichmay be demineralized cortical bone. According to some such embodiments,the at least one growth factor is fibroblast growth factor-2 (FGF-2).According to some such embodiments, the at least one growth factor isfibroblast growth factor-5 (FGF-5). According to some such embodiments,the at least one growth factor is insulin-like growth factor 1 (IGF-1).According to some such embodiments, the at least one growth factor istransforming growth factor beta (TGF-β). According to some suchembodiments, the at least one growth factor is bone morphogenicprotein-2 (BMP-2). According to some such embodiments, the at least onegrowth factor is bone morphogenic protein-7 (BMP-7). According to somesuch embodiments, the at least one growth factor is platelet-derivedgrowth factor (PDGF). According to some such embodiments, the at leastone growth factor is vascular endothelial growth factor (VEGF).According to some such embodiments, the at least one growth factor isneural epidermal growth-factor-like 1 (NELL-1). According to anotherembodiment, the rinsed osteoconductive matrix particles are supplementedwith at least one cytokine.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to oneembodiment, the at least one growth-inductive component is endogenous tothe at least one growth-conductive matrix. According to one embodiment,the tissuegenic cells adherent to and resident in the endogenous milieuof the growth conductive matrix secrete the at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductive component is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells. According to one embodiment, the at leastone growth-inductive component comprises a demineralized bone matrix,which may be demineralized cortical bone.

Processing Step (iii): Processing a Second Growth-Conductive MatrixComprising a Decellularized Growth-Conductive Matrix to Generate aPlurality of Pieces

According to some embodiments, processing step (iii) comprises steps:(1) preparing the at least one growth-conductive matrix derived from atissue so as to yield a plurality of growth-conductive matrix pieces;(2) soaking the plurality of growth-conductive matrix pieces of (1); and(3) decellularizing the plurality of growth-conductive matrix pieces of(2) to generate a plurality of decellularized growth-conductive matrixpieces.

According to some embodiments, preparing step (1) comprises mincing thetissue. According to some embodiments, preparing step (1) comprisescutting the tissue. According to some embodiments, preparing step (1)comprises slicing the tissue. According to some embodiments, preparingstep (1) comprises milling the tissue. According to some embodiments,procuring step (1) comprises homogenizing the tissue.

According to some embodiments, processing step (iii) is at a temperatureof about 25° C. According to some embodiments, processing step (iii) isat a temperature of about 4° C. to about 10° C. According to someembodiments, processing step (iii) is at an ambient temperature.According to some embodiments, preparing step (1) is at a temperature ofabout 25° C. According to some embodiments, preparing step (1) is at atemperature of about 4° C. to about 10° C. According to someembodiments, preparing step (1) is at an ambient temperature. Accordingto some embodiments, soaking step (2) is at a temperature of about 25°C. According to some embodiments, soaking step (2) is at a temperatureof about 4° C. to about 10° C. According to some embodiments, soakingstep (2) is at an ambient temperature. According to some embodiments,decellularizing step (3) is at a temperature of about 25° C. Accordingto some embodiments, decellularizing step (3) is at a temperature ofabout 4° C. to about 10° C. According to some embodiments,decellularizing step (3) is at an ambient temperature.

According to some embodiments, the plurality of growth-conductive matrixpieces of step (1) comprises a plurality of matrix particles. Accordingto some embodiments, the plurality of growth-conductive matrix pieces ofstep (1) comprises a plurality of matrix slices. According to someembodiments, the plurality of growth-conductive matrix pieces of step(1) comprises a plurality of matrix sheets.

According to some embodiments, the plurality of growth-conductive matrixpieces can be of virtually any shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a similar shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of a circular shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a square shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of a polygonal shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a rectangular shape.According to some embodiments, the plurality of growth-conductive matrixpieces are of a triangular shape. According to some embodiments, theplurality of growth-conductive matrix pieces are of a octagonal shape.According to some embodiments, the plurality of growth-conductive matrixare of an irregular shape. According to some embodiments, the pluralityof growth-conductive matrix pieces are of an elongated shape (e.g., as afiber). According to some embodiments, the plurality ofgrowth-conductive matrix pieces are of an amorphous shape.

According to some embodiments, the plurality of growth-conductive matrixpieces comprises at least one growth-conductive matrix piece whoselongest dimension is of about 10 μm to about 20 cm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 10μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 20 μm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 30 μm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about40 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 50 μm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 100 μm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about150 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a osteocondgrowth-conductive matrixpiece whose longest dimension is of about 200 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 250μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 300 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 350μm. According to some such embodiments, the at least growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 400 μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 450 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 500μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 550 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 600μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 650 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 700μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 750 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 800μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 850 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 900μm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 950 μm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is in the rangeof about 100 μm to about 1000 μm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 1 mm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 2 mm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 3 mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 4 mm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 5 mm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 6 mm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 7 mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 8 mm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 9 mm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 10mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is in the range of about 1 mm to about 10 mm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 50 mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 100 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 200mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 300 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 400mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 500 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 600mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 700 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 800mm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 900 mm. According to some suchembodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 1 cm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 2 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 3 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 4 cm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 5 cm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 6 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 7 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 8 cm. According to somesuch embodiments, the at least one growth-conductive matrix piece is agrowth-conductive matrix piece whose longest dimension is of about 9 cm.According to some such embodiments, the at least one growth-conductivematrix piece is a growth-conductive matrix piece whose longest dimensionis of about 10 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 11 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 12 cm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about13 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 14 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 15 cm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about16 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 17 cm. According to some such embodiments,the at least one growth-conductive matrix piece is a growth-conductivematrix piece whose longest dimension is of about 18 cm. According tosome such embodiments, the at least one growth-conductive matrix pieceis a growth-conductive matrix piece whose longest dimension is of about19 cm. According to some such embodiments, the at least onegrowth-conductive matrix piece is a growth-conductive matrix piece whoselongest dimension is of about 20 cm.

According to some embodiments, the plurality of separatedgrowth-conductive matrix pieces can be of any form. According to someembodiments, the plurality of separated growth-conductive matrix piecescomprises a plurality of sheets. According to some embodiments, theplurality of separated growth-conductive matrix pieces comprises apowder form. According to some embodiments, the plurality of separatedgrowth-conductive matrix pieces comprises a slurry form. According tosome embodiments, the plurality of separated growth-conductive matrixpieces comprises a three-dimensional form. According to someembodiments, the three-dimensional form is selected from the groupconsisting of a block, a dowel, a sheet, and a combination thereof.According to some such embodiments, the three-dimensional form comprisesa block. According to some such embodiments, the three-dimensional formcomprises a dowel. According to some such embodiments, thethree-dimensional form comprises a sheet.

According to some embodiments, soaking step (2) comprises soaking theplurality of growth-conductive matrix pieces of (1) with a liquid.According to some embodiments, soaking step (2) comprises soaking theplurality of growth-conductive matrix pieces of (1) with a liquid,wherein the liquid is a buffer. According to some embodiments, soakingstep (2) comprises soaking the plurality of growth-conductive matrixpieces of (1) with a buffered isotonic solution. According to someembodiments, soaking step (2) comprises soaking of the plurality ofgrowth-conductive matrix pieces of (1) with phosphate buffered saline(PBS). According to some embodiments, soaking step (2) comprises soakingthe plurality of growth-conductive matrix pieces of (1) with trisbuffered saline (TBS). According to some embodiments, soaking step (2)comprises soaking the plurality of growth-conductive matrix pieces of(1) with deionized water.

According to some embodiments, decellularizing step (3) comprises steps:(1) delipidating the plurality of growth-conductive matrix pieces; and(2) disinfecting the plurality of growth-conductive matrix pieces; togenerate a plurality of pieces comprising a decellularized tissuederived growth-conductive matrix.

According to some embodiments, decellularizing step (3) furthercomprises step (4) lyophilizing the decellularized tissue derivedgrowth-conductive matrix and exposing it to a freezer mill to generate aparticularized tissue derived matrix powder. According to someembodiments, decellularizing step (3) further comprises step (4)homogenizing the decellularized tissue derived growth-conductive matrixto generate a decellularized tissue derived matrix paste. According tosome embodiments, decellularizing step (3) further comprises step (4)homogenizing the decellularized tissue derived growth-conductive matrixto generate a decellularized tissue derived matrix slurry. According tosome embodiments, decellularizing step (3) further comprises step (4)homogenizing the decellularized tissue derived growth-conductive matrixand a further step (5) lyophilizing the decellularized tissue derivedgrowth-conductive matrix to generate a three dimensional decellularizedtissue derived matrix. According to some embodiments, decellularizingstep (3) further comprises a step (4) lyophilizing the decellularizedtissue derived growth-conductive matrix to generate a decellularizedtissue derived matrix sheet.

According to another embodiment, the method of fabricating an implantfurther comprises step (vii) supplementing the plurality ofgrowth-conductive matrix pieces. According to some such embodiments, theat least one growth-inductive component comprises at least onegrowth-inductive factor. According to some such embodiments, the atleast one growth-inductive factor comprises at least one growth factor.According to some such embodiments, the at least one growth factor isfibroblast growth factor-2 (FGF-2). According to some such embodiments,the at least one growth factor is fibroblast growth factor-5 (FGF-5).According to some such embodiments, the at least one growth factor isinsulin-like growth factor 1 (IGF-1). According to some suchembodiments, the at least one growth factor is transforming growthfactor beta (TGF-β). According to some such embodiments, the at leastone growth factor is bone morphogenic protein-2 (BMP-2). According tosome such embodiments, the at least one growth factor is bonemorphogenic protein-7 (BMP-7). According to some such embodiments, theat least one growth factor is platelet-derived growth factor (PDGF).According to some such embodiments, the at least one growth factor isvascular endothelial growth factor (VEGF). According to some suchembodiments, the at least one growth factor is neural epidermalgrowth-factor-like 1 (NELL-1). According to another embodiment, therinsed osteoconductive matrix particles are supplemented with at leastone cytokine.

According to some such embodiments, the at least one growth-inductivecomponent comprises a demineralized bone matrix, which may bedemineralized cortical bone. According to some embodiments, thedemineralized bone matrix is provided as a plurality of pieces ofdemineralized bone matrix derived from a single piece of demineralizedbone. According to some embodiments, the demineralized bone matrix isprovided as a plurality of pieces of demineralized bone matrix derivedfrom a plurality of pieces of bone tissue.

According to some embodiments, the pieces of bone tissue that are to bedemineralized have a longest dimension (which is used herein as anequivalent term to “maximum dimension”) that is measurable prior todemineralization of the pieces of bone. All such longest dimensionsrelated in this paragraph are the longest dimension of a piece of bonetissue prior to demineralization. According to some embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 10 μm to about 20 cm.According to some such embodiments, the plurality of pieces of bonetissue includes a piece of bone tissue whose longest dimension is ofabout 10 μm. According to some such embodiments, the plurality of piecesof bone tissue includes a piece of bone tissue whose longest dimensionis of about 20 μm. According to some such embodiments, the plurality ofpieces of bone tissue includes at least one piece of bone tissue whoselongest dimension is of about 30 μm. According to some such embodiments,the plurality of pieces of bone tissue includes at least one piece ofbone tissue whose longest dimension is of about 40 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 50μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 100 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 150 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 200μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 250 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 300 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 350μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 400 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 450 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 500μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 550 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 600 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 650μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 700 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 750 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 800μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 850 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 900 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 950μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is in the range of about 100 μm to about 1000 μm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose longest dimension is of about 1mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 2 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 3 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 4 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 5 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 6 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 7 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 8 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 9 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 10 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is in the range of about 1 mm to about 10 mm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose longest dimension is of about 50mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 100 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 200 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 300mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 400 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 500 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 600mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 700 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 800 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose longest dimension is of about 900mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 1 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 2 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 3 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 4 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 5 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 6 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 7 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 8 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 9 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 10 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 11 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 12 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 13 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 14 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 15 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 16 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 17 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose longest dimension is of about 18 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose longestdimension is of about 19 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose longest dimension is of about 20 cm.

According to some embodiments, the pieces of bone tissue that are to bedemineralized have a shortest dimension (which is used herein as anequivalent term to “minimum dimension”) that is measurable prior todemineralization of the pieces of bone. All such shortest dimensionsrelated in this paragraph are the shortest dimension of a piece of bonetissue prior to demineralization. According to some embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 10 μm to about 20 cm.According to some such embodiments, the plurality of pieces of bonetissue includes a piece of bone tissue whose shortest dimension is ofabout 10 μm. According to some such embodiments, the plurality of piecesof bone tissue includes a piece of bone tissue whose shortest dimensionis of about 20 μm. According to some such embodiments, the plurality ofpieces of bone tissue includes at least one piece of bone tissue whoseshortest dimension is of about 30 μm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 40 μm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 50 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 100 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 150μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 200 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 250 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 300μm. According to some embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is in the range of about 5 μm to about 300 μm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose shortest dimension is of about350 μm. According to some such embodiments, the plurality of pieces ofbone tissue includes at least one piece of bone tissue whose shortestdimension is of about 400 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 450 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 500μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 550 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 600 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 650μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 700 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 750 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 800μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 850 μm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 900 μm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 950μm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is in the range of about 100 μm to about 1000 μm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose shortest dimension is of about 1mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 2 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 3 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 4 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 5 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 6 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 7 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 8 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 9 mm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 10 mm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is in the range of about 1 mm to about 10 mm. According tosome such embodiments, the plurality of pieces of bone tissue includesat least one piece of bone tissue whose shortest dimension is of about50 mm. According to some such embodiments, the plurality of pieces ofbone tissue includes at least one piece of bone tissue whose shortestdimension is of about 100 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 200 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 300mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 400 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 500 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 600mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 700 mm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 800 mm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 900mm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 1 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 2 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 3 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 4 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 5 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 6 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 7 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 8 cm. According to some suchembodiments, the plurality of pieces of bone tissue includes at leastone piece of bone tissue whose shortest dimension is of about 9 cm.According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 10 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 11 cm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 12cm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 13 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 14 cm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 15cm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 16 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 17 cm. According to somesuch embodiments, the plurality of pieces of bone tissue includes atleast one piece of bone tissue whose shortest dimension is of about 18cm. According to some such embodiments, the plurality of pieces of bonetissue includes at least one piece of bone tissue whose shortestdimension is of about 19 cm. According to some such embodiments, theplurality of pieces of bone tissue includes at least one piece of bonetissue whose shortest dimension is of about 20 cm.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to oneembodiment, the at least one growth-inductive component is endogenous tothe at least one growth-conductive matrix. According to one embodiment,the tissuegenic cells adherent to and resident in the endogenous milieuof the growth conductive matrix secrete the at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductivecomponent is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells. According to one embodiment, the at leastone growth-inductive component comprises a demineralized bone matrix,which may be demineralized cortical bone.

Seeding Step (iv): Seeding the Decellularized Growth-Conductive MatrixPieces of (iii) with the Plurality of at Least One Viable IsolatedPopulation of Tissuegenic Cells of (ii) to Form a Plurality of ReseededGrowth-Conductive Matrix Pieces, where the at Least One ViablePopulation of Tissuegenic Cells is Caused to be in Contact with theGrowth-Conductive Matrix

According to some embodiments, seeding step (iv) comprises the steps:(1) providing a suspension comprising a plurality of the at least oneviable isolated population of tissuegenic cells; (2) admixing a portionof the suspension comprising a plurality of the at least one viableisolated population of tissuegenic cells with a plurality of piecescomprising a decellularized tissue derived growth-conductive matrix; (3)bringing the at least one viable isolated population of tissuegeniccells into contact with the matrix; (4) rinsing the at least one viablepopulation of tissuegenic cells caused to be in contact with a pluralityof pieces comprising the decellularized tissue derived matrix; to yieldthe implant.

According to one embodiment, the incubating step comprises placing theplurality of pieces comprising a decellularized tissue derived matrix ata temperature of 37° C. for 24 hours. According to one embodiment, theincubating step comprises placing the plurality of pieces comprising adecellularized tissue derived matrix at a temperature of 37° C. for 24hours under static seeding conditions. According to one embodiment, theincubating step comprises placing the plurality of pieces comprising adecellularized tissue derived matrix at a temperature of 37° C. for 24hours under dynamic seeding condition with gentle agitation.

According to one embodiment, the rinsing step comprises washing with aliquid to remove non-adhering cells. According to some embodiments, theliquid comprises a buffered isotonic solution. According to someembodiments, the liquid comprises a buffered salt solution. According tosome embodiments, the buffered salt solution comprises a PBS solution.According to some embodiments, the buffered salt solution comprises aTBS solution.

According to another embodiment, the method of fabricating an implantfurther comprises step (vii) supplementing the implant of step (iv).According to some such embodiments, the at least one growth-inductivecomponent comprises at least one growth-inductive factor. According tosome such embodiments, the at least one growth-inductive factorcomprises at least one growth factor. According to some suchembodiments, the at least one growth-inductive component comprises ademineralized bone matrix, which may be demineralized cortical bone.According to some such embodiments, the at least one growth factor isfibroblast growth factor-2 (FGF-2). According to some such embodiments,the at least one growth factor is fibroblast growth factor-5 (FGF-5).According to some such embodiments, the at least one growth factor isinsulin-like growth factor 1 (IGF-1). According to some suchembodiments, the at least one growth factor is transforming growthfactor beta (TGF-β). According to some such embodiments, the at leastone growth factor is bone morphogenic protein-2 (BMP-2). According tosome such embodiments, the at least one growth factor is bonemorphogenic protein-7 (BMP-7). According to some such embodiments, theat least one growth factor is platelet-derived growth factor (PDGF).According to some such embodiments, the at least one growth factor isvascular endothelial growth factor (VEGF). According to some suchembodiments, the at least one growth factor is neural epidermalgrowth-factor-like 1 (NELL-1). According to another embodiment, therinsed osteoconductive matrix particles are supplemented with at leastone cytokine.

According to one embodiment, the at least one growth-inductive componentis tissue-derived. According to one embodiment, the at least onegrowth-inductive component comprises inducible pluripotent stem cells(iPSCs). According to one embodiment, the at least one growth-inductivecomponent originates from a component of the tissue-derivedgrowth-inductive component other than cells. According to oneembodiment, the at least one growth-inductive component is endogenous tothe at least one growth-conductive matrix. According to one embodiment,the tissuegenic cells adherent to and resident in the endogenous milieuof the growth conductive matrix secrete the at least onegrowth-inductive component. According to one embodiment, the at leastone growth-inductivecomponent is exogenous to the at least onegrowth-conductive matrix. According to one embodiment, thegrowth-inductive component comprises a growth medium derived fromexpanded tissuegenic cells. According to one embodiment, the at leastone growth-inductive component comprises a demineralized bone matrix,which may be demineralized cortical bone.

3. An Implant Fabricated by the Methods Described

According to another aspect, the described invention provides an implantfabricated by a method comprising steps:

(a) providing at least one growth-conductive matrix or at least onefragment thereof, wherein the growth-conductive matrix comprises atleast one viable population of tissuegenic cells endogenous to thetissue, wherein the tissuegenic cells are adherent to and resident inthe endogenous milieu of the growth-conductive matrix;

(b) separating the at least one growth-conductive matrix of (a) togenerate a plurality of separated matrix pieces comprising the at leastone viable population of tissuegenic cells caused to be in contact withand resident in the endogenous milieu of the growth-conductive matrixwherein the relative frequency of a stem cell subtype in the at leastone viable tissuegenic cell population of step (b) is of a similarrelative frequency as found in the growth-conductive matrix of step (a);

(c) rinsing the plurality of separated matrix pieces of (b) comprisingthe at least one viable population of tissuegenic cells caused to be incontact with and resident in the endogenous milieu of thegrowth-conductive matrix of (b) to form a plurality of rinsed separatedmatrix pieces comprising the at least one viable population oftissuegenic cells caused to be in contact with and resident in the theendogenous milieu of the growth-conductive matrix of (b), wherein arelative frequency of a stem cell subtype in the at least one viabletissuegenic cell population of step (c) is of a similar relativefrequency to that found in the growth-conductive matrix of step (a);

(d) collecting the plurality of rinsed separated matrix pieces of (c)comprising the at least one viable population of tissuegenic cellscaused to be in contact with and resident in the endogenous milieu ofthe growth-conductive matrix of (c) wherein the at least one viabletissuegenic cell population is of a similar relative frequency to thatfound in the growth-conductive matrix of step (a);

(e) packaging the plurality of collected rinsed separated matrix piecesof (d) comprising at least one viable population of tissuegenic cellscaused to be in contact with and resident in the endogenous milieu ofthe growth-conductive matrix of (c) wherein the at least one viabletissuegenic cell population is of a similar relative frequency as foundin the growth-conductive matrix of step (a) to form the implant.

According to another embodiment, the described invention provides animplant fabricated by a method comprising steps:

(i) providing at least one first growth-conductive matrix or at leastone fragment thereof, derived from human tissue, wherein thegrowth-conductive matrix comprises at least one viable population oftissuegenic cells endogenous to the tissue, wherein the tissuegeniccells are adherent to and resident in the endogenous milieu of thegrowth-conductive matrix;

(ii) isolating from the at least one growth-conductive matrix of (i) aplurality of the at least one viable isolated population of tissuegeniccells;

(iii) processing a second growth-conductive matrix comprising adecellularized growth-conductive matrix to generate a plurality ofpieces;

(iv) seeding the decellularized growth-conductive matrix pieces of (iii)with the plurality of at least one viable isolated population oftissuegenic cells of (ii) to form a plurality of reseededgrowth-conductive matrix pieces, where the at least one viablepopulation of tissuegenic cells is caused to be in contact with thegrowth-conductive matrix;

(v) collecting the plurality of reseeded growth-conductive matrix piecesof (iv) comprising the at least one viable population of tissuegeniccells caused to be in contact with or in contact with thegrowth-conductive matrix of (iv), wherein the at least one viabletissuegenic cell population is of a relative frequency substantiallysimilar to that found in the growth-conductive matrix of step (i);

(vi) packaging the plurality of collected reseeded growth-conductivematrix pieces of (v) comprising the at least one viable population oftissuegenic cells caused to be in contact with or in contact with thegrowth-conductive matrix of (iv), wherein the at least one viabletissuegenic cell population is of a relative frequency substantiallysimilar to that found in the growth-conductive matrix of step (i) toform the implant.

A Fabricated Orthopedic Implant

According to one embodiment, the described invention provides anorthopedic implant fabricated by a method comprising steps:

(a) providing at least one growth-conductive matrix or at least onefragment thereof, wherein the growth-conductive matrix comprises atleast one viable population of tissuegenic cells caused to be in contactwith and resident in the growth-conductive matrix;

(b) milling the growth-conductive matrix of (a) to generate a pluralityof milled matrix particles comprising the at least one viable populationof tissuegenic cells caused to be in contact with and resident in thegrowth-conductive matrix, wherein a relative frequency of a cell subtypein the at least one viable tissuegenic cell population of step (b) is ofa relative frequency substantially similar to that found in thegrowth-conductive matrix of step (a);

(c) rinsing the plurality of milled matrix particles of (b) comprisingthe at least one viable population of tissuegenic cells caused to be incontact with and resident in the growth-conductive matrix of (b) to forma plurality of rinsed milled matrix particles comprising the at leastone viable population of tissuegenic cells caused to be in contact withand resident in the growth-conductive matrix of (b), wherein a relativefrequency of a cell subtype in the at least one viable tissuegenic cellpopulation of step (c) is of a relative frequency substantially similarto that found in the growth-conductive matrix of step (a);

(d) collecting the plurality of rinsed matrix particles comprising atleast one viable population of tissuegenic cells caused to be in contactwith and resident in the growth-conductive matrix of (c) wherein the atleast one viable tissuegenic cell population is of a relative frequencysubstantially similar to that found in the growth-conductive matrix ofstep (a), step (b) or step (c); to form the orthopedic implant.

The method of fabricating an implant may include the addition of ademineralized bone matrix, which may be demineralized cortical bone.According to some embodiments, the implant is an implantable compositioncomprising a growth-conductive matrix (e.g., an osteoconductive matrixderived from a bone tissue), a viable population of tissuegenic cells(e.g., osteogenic cells), and an osteoinductive matrix (e.g., ademineralized bone matrix). In an embodiment, a osteoinductive matrix(e.g., a second demineralized bone matrix) is provided separately fromthe aforesaid implantable composition for addition to the implantablecomposition at a later time.

4. Uses of the Implants of the Described Invention for Tissue Repair

According to another aspect, the implant of the described invention canbe used for a tissue engineering application. Tissue engineering is theapplication of principles and methods of engineering and life sciencestoward a fundamental understanding and development of biologicalsubstitutes to restore, maintain and improve human tissue functions.According to one embodiment, the tissue engineering application is asurgical application. According to one embodiment, the tissueengineering application is a nonsurgical application.

According to one embodiment, the implant can be used for tissue repair.According to one such embodiment, the implant is an orthopedic implant.According to one embodiment, a method for treating a bony defect at adefect site in a subject in need thereof comprises the steps:

(a) providing an orthopedic implant comprising

-   -   (i) a plurality of pieces comprising at least one tissue-derived        growth-conductive matrix; and    -   (ii) at least one viable population of tissuegenic cells        adherent to and resident in an endogenous milieu of the        growth-conductive matrix;

(b) implanting the orthopedic implant at the defect site; and

(c) filling the bony defect.

According to another embodiment, the implant is an implantablecomposition comprising a growth-conductive matrix (e.g., anosteoconductive matrix derived from bone), a viable population oftissuegenic cells (e.g., osteogenic cells), and an osteoinductive matrix(e.g., demineralized bone matrix). In some such embodiments, thecomposition is applied to the bone defect so as to fill the bone defect.In some such embodiments, the composition is applied with a spatula. Insome such embodiments, the composition is injected into the bony defect.In another embodiment, a second osteoinductive matrix (e.g., a seconddemineralized bone matrix) is provided separately from the aforesaidimplantable composition, and is mixed with the implantable compositionat the surgical location, before application to the bone defect.

According to one embodiment of the method, the bony defect resulted fromtumor surgery. According to another embodiment, the bony defect resultedfrom a traumatic injury. According to another embodiment, the bonydefect resulted from a congenital skeletal abnormality. According toanother embodiment, the bony defect resulted from a fracture. Accordingto another embodiment, the bony defect resulted from a spinalarthrodesis. According to another embodiment, the bony defect resultedfrom a joint replacement.

According to one embodiment, the tissue to be repaired using the implantis selected from the group consisting of an adipose tissue, an amniontissue, an artery tissue, a bone tissue, a cartilage tissue, a choriontissue, a colon tissue, a dental tissue, a dermal tissue, a duodenaltissue, an endodermal tissue, an epithelial tissue, a fascial tissue, agastrointestinal tissue, a growth plate tissue, an intervertebral disctissue, an intestinal mucosal tissue, an intestinal serosal tissue, akidney tissue, a ligament tissue, a liver tissue, a lung tissue, amammary tissue, a meniscal tissue, a muscle tissue, a nerve tissue, anovarian tissue, a pancreatic tissue, a parenchymal organ tissue, apericardial tissue, a periosteal tissue, a peritoneal tissue, aplacental tissue, a reproductive epithelial tissue, a respiratoryepithelial tissue, a skin tissue, a spleen tissue, a stomach tissue, asynovial tissue, a tendon tissue, a testes tissue, an umbilical cordtissue, a urological tissue, a vascular tissue, a vein tissue, and acombination thereof.

According to one embodiment, the implant can be used in repairingadipose tissue. According to one embodiment, the implant can be used inrepairing amnion tissue. According to one embodiment, the implant can beused in repairing artery tissue. According to one embodiment, theimplant can be used in repairing bone tissue. According to oneembodiment, the implant can be used in repairing cartilage tissue.According to one embodiment, the implant can be used in repairingchorion tissue. According to one embodiment, the implant can be used inrepairing colon tissue. According to one embodiment, the implant can beused in repairing dental tissue. According to one embodiment, theimplant can be used in repairing dermal tissue. According to oneembodiment, the implant can be used in repairing duodenal tissue.According to one embodiment, the implant can be used in repairingendothelial tissue. According to one embodiment, the implant can be usedin repairing epithelial tissue. According to one embodiment, the implantcan be used in repairing fascial tissue. According to one embodiment,the implant can be used in repairing gastrointestinal tissue. Accordingto one embodiment, the implant can be used in repairing growth platetissue. According to one embodiment, the implant can be used inrepairing intervertebral disc tissue. According to one embodiment, theimplant can be used in repairing intestinal mucosal tissue. According toone embodiment, the implant can be used in repairing intestinalscelerosal tissue. According to one embodiment, the implant can be usedin repairing kidney tissue. According to one embodiment, the implant canbe used in repairing ligament tissue. According to one embodiment, theimplant can be used in repairing liver tissue. According to oneembodiment, the implant can be used in repairing lung tissue. Accordingto one embodiment, the implant can be used in repairing mammary tissue.According to one embodiment, the implant can be used in repairingmeniscal tissue. According to one embodiment, the implant can be used inrepairing muscle tissue. According to one embodiment, the implant can beused in repairing nerve tissue. According to one embodiment, the implantcan be used in repairing ovarian tissue. According to one embodiment,the implant can be used in repairing pancreatic tissue. According to oneembodiment, the implant can be used in repairing parenchymal organtissue. According to one embodiment, the implant can be used inrepairing pericardial tissue. According to one embodiment, the implantcan be used in repairing periosteal tissue. According to one embodiment,the implant can be used in repairing peritoneal tissue. According to oneembodiment, the implant can be used in repairing placental tissue.According to one embodiment, the implant can be used in repairingreproductive epithelial tissue. According to one embodiment, the implantcan be used in repairing respiratory epithelial tissue. According to oneembodiment, the implant can be used in repairing skin tissue. Accordingto one embodiment, the implant can be used in repairing spleen tissue.According to one embodiment, the implant can be used in repairingstomach tissue. According to one embodiment, the implant can be used inrepairing synovial tissue. According to one embodiment, the implant canbe used in repairing tendon tissue. According to one embodiment, theimplant can be used in repairing testes tissue. According to oneembodiment, the implant can be used in repairing umbilical cord tissue.According to one embodiment, the implant can be used in repairingurological tissue. According to one embodiment, the implant can be usedin repairing vascular tissue. According to one embodiment, the implantcan be used in repairing vein tissue.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperatures, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees centigrade,and pressure is at or near atmospheric.

I. Source Tissue Bone Example 1 Orthopedic Implant Fabrication

Human ilium is recovered aseptically from deceased donors between theages of 18 and 45 years of age within 24 hours post-mortem to yieldilium tissue. The ilium tissue is stored at 4° C. until ready forprocessing. Generally, tissue processing commences within 72 hourspost-mortem. The ilium tissue is exposed to a bioburden reducer togenerate preprocessed ilium tissue. The preprocessed ilium tissue issubjected to three to four 5 minute soaks with agitation in a bufferedisotonic solution (e.g., PBS (0.01 M, pH 7.4)). The preprocessedagitated ilium tissue then is debrided of all soft tissue and cut intostrips of approximately 3×3 cm. The crest is cut off and used for othergrafts, and the acetabulum is discarded. The strips are milled intoparticles approximately 2 mm in diameter or less using a mill. Allcomponents of the mill that come in contact with the tissue are chilledat 4° C. until use. In some embodiments, the preprocessed ilium tissueis crushed to form fragments or a powder.

The particles then are subjected to a series of rinses with cold PBS,followed by a rinse in acetic acid solution for 5 minutes and thenfollowed again by a series of rinses with cold PBS. The pH of therinseate is at or near physiological pH at the end of the rinses.

Prior to packaging, demineralized bone matrix or another osteoinductivematrix may be added to the tissue to enhance the osteoinductive natureof the tissue form. The demineralized bone matrix may be provided asparticles, which may be prepared from particles of bone, or as elongatedparticles (e.g., as in fibers), or in some other form.

The tissue is placed in a cryopreservation solution and cryopreservedusing a controlled rate freezer.

In another embodiment, a second demineralized bone matrix, which may beelongated particles (e.g., as in fibers) of demineralized bone matrix issupplied separately from the implant composition that includesbone-derived particles and demineralized bone matrix, to be mixed withthe implant composition at the surgical location. In an example of suchan embodiment, a first container of the implant compositions and asecond container of the second demineralized bone matrix are providedtogether as a kit. Implements for mixing the contents of the first andsecond containers, and for implanting the resulting mixture in a bonedefect may be included in the kit.

It should be understood by those skilled in the art that manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to improve, forexample, but not limited to, efficiency, and yield.

Example 2 Identification of Osteogenic Cells

Putative osteogenic cells are assayed in vitro to confirm their identityas MSCs or as osteoprogenitor cells, by using techniques, well-known inthe art, including but not limited to oil red O staining assay, VonKossa staining assay, colony forming unit fibroblast assay, alkalinephosphatase assay, etc.

Example 2.1 Identification of Mesenchymal Stem Cells

Mesenchymal stem cells initially are identified by phase microscopy.Explanted putative MSCs then are assayed in vitro to determine theirability to differentiate into 1) an osteogenic lineage, 2) an adipogeniclineage, and 3) a chondrogenic lineage.

Example 2.1.1 Lipid Accumulation

Preparation of Solutions

5% Oil Red O (Sigma) stock solutions in isopropanol are prepared. 6 mlof stock is added to 4 ml double distilled H₂O, mixed well and filtered.Generally, 500 μl to 1 ml are required per well of a 24 well plate.

Oil Red Assay

Lipid accumulation in fully differentiated cells is analyzed. First,medium is aspirated carefully from each well so as not to aspirate thecells. Second, plates are rinsed with PBS (1×), cells are fixed bycovering with buffered formalin, and then plates are allowed to sit forat least 15 minutes at room temperature. Third, fixative agent isremoved, the plates are rinsed 3 times with PBS (1×), and Oil Red isthen added. The plates are allowed to sit at room temperature(approximately 25° C.) for at least 1 hour, and then are rinsed 3 timeswith double distilled H₂O (1 ml) to remove excess stain and anyprecipitate that forms. Cell nuclei are stained with Hematoxylinsolution (0.5 ml) for 5 to 15 minutes, then rinsed 3 times with doubledistilled H₂O, and allowed to air dry.

The Oil Red O solution stains adipocytes containing lipid droplets red;hematoxylin stains the cell nucleus black/blue.

Example 2.1.2 Determination of Mineralization

Von Kossa Staining

Mesenchymal stem cells that have been induced to follow an osteogeniclineage are stained to determine whether the cultures have mineralized.A 2% solution of silver nitrate (w/v) is prepared, avoiding lightexposure. Cultures are rinsed twice with cold Tyrode's balanced saltsolution (or Hank's balanced salt solution (HBSS)), fixed with 10%buffered formalin phosphate for 30 minutes (if the cultures werepreviously stained with fast violet, the plates are kept covered infoil). The culture is rinsed with distilled water, 2% silver nitrate(volume similar to normal volume used for cell culture media for theflask/wells being stained) is added, and allowed to incubate for 10minutes while covered in foil. The staining solution is removed andcultures are rinsed 3 times with distilled water, leaving the water fromthe final rinse on the cultures. The cultures are placed on a bright,white background and exposed to bright light for 15 minutes, the watercovering the cells is removed and the cultures are rinsed twice withwater. The cultures are dehydrated with 100% ethanol for 1 minute, andallowed to air dry. Areas that are stained brown or black indicatemineralization. The cultures can be examined under the microscope toobserve any diffuse staining.

Example 2.1.3 Mesenchymal Stem Cell Identification

Colony Forming Unit-Fibroblast Assay

Mesenchymal stem cells (MSCs) are identified using a colony formingunit-fibroblast (CFU-F) assay following the manufacturer's instructions(Stem Cell Technologies).

Culture Set-Up

Briefly, the CFU-F assay culture set-up is as follows: 1) MesenchymalStem Cell Stimulatory Supplements are thawed at room temperature or 4°C. overnight. The entire contents of the Mesenchymal StimulatorySupplements are added to MesenCult Basal Medium for Human MesenchymalStem Cells and mixed thoroughly. This is now referred to as completemedium; 2) the normal bone marrow samples of red blood cells are lysed(i.e., using an ammonium chloride solution) or a mononuclear cellfraction is prepared by Ficoll-Paque density gradient separation; 3)cells are washed by adding 10 ml of PBS containing 2% FBS to the cellpellet. The cells are centrifuged at 1200 rpm for 10 minutes at 20° C.,the supernatant is removed and the cells resuspended in MesenCultcomplete medium; 4) 1.0 ml, 0.5 ml and 0.25 ml of the cell stocksolution is added to separate 100 mm tissue culture treated dishes (orT-25 cm² tissue culture flasks) to yield final cell concentrations of2×10⁶ cells, 1×10⁶ cells, and 0.5×10⁶ cells respectively in a totalvolume of 10 ml of MesenCult medium. These concentrations will ensurethat the resulting numbers of colonies can be scored, as there aredifferences in the proliferative potential of CFU-F from various bonemarrow samples; 6) the 100 mm dishes (or T-25 cm² tissue culture flasks)are placed into a 37° C. humidified incubator with 5% CO₂ in airand >95% humidity for 14 days. Maximum colony size and numbers typicallyare observed at this time.

Staining the CFU-F (Stromal Stem Cells) Colonies

The CFU-F colonies are stained as follows. Briefly, the media from thetissue culture dishes is removed to T-25 cm² tissue culture flasks anddiscarded. The culture dishes or flasks are washed twice using PBS (toremove any remaining medium) and the PBS from the two washes isdiscarded. Next, 5 ml of methanol is added and the culture dishes orflasks are allowed to air dry at room temperature. The methanol isdiscarded and 5 ml of Giemsa staining solution is added to each culturedish or flask for five minutes. The Giemsa staining solution is removedand the culture dishes or flasks rinsed with distilled water. Thedistilled water is discarded and the tissue culture dishes or flasks areallowed to dry at room temperature.

Scoring Procedure

Typically, the CFU-F colonies are between 1 mm and 8 mm in diameter andmay be scored macroscopically. By confirming that there are as manycolonies when cells are plated at 2×10⁶/flask as compared to1.0×10⁶/flask and that there should be twice as many colonies when cellsare plated at 1.0×10⁶/flask as compared to the 0.5×10⁶/flask, it ispossible to ensure that there is a linear relationship between the cellnumbers that are plated and the resulting colony numbers.

Example 2.1.4 Osteogenic Stem Cell Identification

Cells are assayed for their ability to differentiate into osteogenicprogenitors using commercially available kits from, for example, StemCell Technologies, Inc.

Medium

Fresh “complete medium” is prepared weekly for the maintenance ofcultures along the osteogenic lineage. MesenCult Basal Medium is storedat 4° C. in 10×45 ml aliquots; Osteogenic Stimulatory Supplements isused at a final 15% volume and stored at −20° C. in 10×8 ml aliquots;β-glycerophosphate is used at a final concentration of 3.5 mM in humanassays (5.0 mM in rat assays) and is stored at −20° C. in 10×1 mlaliquots; and dexamethasone is used at a final concentration of 10⁻⁸ M.Briefly, the powder is dissolved in a small volume of absolute ethanoland made up with ethanol to a final volume of 25.5 ml, then stored at−20° C. in 500 μl aliquots; ascorbic acid is used at a finalconcentration of 50 μg/ml. The powder is dissolved in 10 ml of MesenCultBasal Medium thereby generating a stock solution of 10 mg/ml, and storedat −20° C. in 10×1 ml aliquots.

Complete Medium

MesenCult Basal Medium (42.5 ml) is pipetted into a 50 ml conical tubeand the following: osteogenic supplements are added (7.5 ml);dexamethasone (10-4 M stock solution, 5 μl); ascorbic acid (10 mg/mlstock solution; 250 μl); β-glycerophosphate (1 M stock solution; 175 μl)if needed. Typically, β-glycerophosphate is added only after there isevidence, by phase microscopy, of cell multilayering.

There are many protocols in the literature for the development ofosteogenic cells from various tissue sources including, but not limitedto, bone marrow, cultured mesenchymal cells, adipose-derived stem cells,and aminiotic epithelial or stromal cells. The protocol below is justone example of a method that supports the growth of osteogenic cellsfrom human bone marrow. The described complete medium supports theproliferation of rat osteogenic cells. The optimal concentration ofβ-glycerophosphate used in these studies is 5 mM.

Protocol

The assay is performed as follows: 1) cancellous bone fragments areprepared by mincing the bone into very small pieces (1-3 mm in size); 2)fragments are flushed with 20-30 ml of PBS and the fragments thenvortexed with another 20-30 ml PBS; 3) the cell suspension is passedthrough a sieve to remove bone fragments; 4) cells are spun down at 400g for 15 minutes; 5) the supernatant is discarded and the cells areresuspended in PBS; 6) cells are placed on Ficoll-Paque and spin at 400g for 25 minutes; 7) the cells at the interface are removed andresuspended in complete medium (without β-glycerophosphate); 8) cellsare seeded in tissue culture treated flasks or plates at a concentrationof 100-200,000 cells per cm²; 9) the cultures are re-fed for the firsttime after 5 days by removing the medium (and non-adherent cells). Thesecan be discarded. The cultures are replenished with fresh completemedium (again without β-glycerophosphate unless cell multi-layering hasbeen noted); and 10) cultures are re-fed every 2-3 days for a minimum ofthree weeks (for rat cultures) or 5 weeks (for human cultures).Detection of osteogenic cells may be determined by tetracycline labelingor von Kossa staining at this time. If unprocessed bone marrow cells areavailable, the bone marrow is diluted 1:3 with PBS/2% FBS and theprocess started at point 6 above.

Example 2.1.5 Determination of Alkaline Phosphatase Activity(Qualitative)

Cultures induced along the osteogenic lineage are stained to visualizeany production of alkaline phosphatase (AP).

Preparation of Solutions

Fast Violet Stock is prepared by i) adding one capsule of Fast Violet to48 ml water; ii) allowing the capsule to soften, then mixing thecontainer occasionally until all the Fast Violet is dissolved; iii)aliquoting into 12 ml units and storing the stocks at 4° C. until readyto use.

Citrate Working Solution is prepared by adding 2 ml citrate concentratedsolution to 98 ml Millipore water.

Citrate Buffered Acetone is prepared by combining Citrate WorkingSolution with acetone to give a solution that is 60% Citrate WorkingSolution and 40% acetone by volume.

Staining Cultures

Cultures are stained as follows: i) growth media is removed from theculture to be stained; ii) the culture is rinsed 2 times with Tyrode'ssalt solution (or equivalent); iii) citrate buffered acetone is addedand allowed to sit for 30 seconds (to fix the cells); iv) cultures arerinsed twice with water (the water from the second rinse is left on theculture until ready to proceed to the next step); v) 0.5 ml NaphtholAS-MX (Sigma) is added to 12 ml of Fast Violet solution (this solutionis light sensitive); vi) the water is removed from cultures and anappropriate volume of Fast Violet/Naphthol solution added to each dishor well (volume will vary with the size of the culture dish or well;generally, the same volume of stain is used as the volume of culturemedium used on the plate); vii) the dish is incubated at roomtemperature (about 25° C.) for 45 minutes (avoid light exposure); andviii) the stain solution is removed and the culture rinsed twice withMillipore water. Areas with purple coloration indicate AP production.The cultures may be stored with water on the growth surface in the darkand stained later with Von Kossa.

Example 3 Characterization of Orthopedic Implant

The orthopedic implant is characterized to determine 1) the cell number(quantification of cells within the bone matrix), 2) cell type(identification of cell populations present in the tissue), 3) cellviability (percentage of viable cells after cryopreservation and thaw),and 4) osteoconductivity of the demineralized component (verification ofBMP-2 content).

Example 3.1 Cell Count and Identification

Histological slides are prepared to quantitate cell numbers and celltypes of the bone matrix. Briefly, tissue sections (5 μm) are cut, thenstained with hematoxylin and eosin (H&E), the MSC marker CD166+, theosteoprogenitor cell marker osteocalcin, and hematopoietic cell markersCD34+ and CD45+. Slides then are quantitated. Quantification can beperformed via microscopic image analysis with commercially availablesystems such as, but not limited to, Image Pro Plus and Aperio (Vista,Calif.).

Stereological methods are used to quantify total cell number and thenumber of MSC and osteoprogenitor cells. Stereology is aninterdisciplinary field that allows for extraction of quantitativeinformation about a three-dimensional material from measurements made ontwo-dimensional planar sections of the material. Stereology utilizesrandom, systematic sampling to provide unbiased and quantitative data.Briefly, conversion of cell number per cm³ of tissue is based on scannedserial tissue sections where slides are overlaid to identify an area ofinterest. A controlled estimation of cell numbers is determined using anOptical Disector. This is an extension of the basic Disector method,which is applied to a thick section using a series, or stack, ofDisectors. Rather than using pairs of physical sections (the basicDisector method), optical sectioning is used by creating focal planeswith a thin depth-of-field through the section. The Optical Disectorbegins with a lookup section at the top of the optical disector and endswith a reference section at the bottom of the optical disector. Thefocal plane is the current reference section. The lookup section isimmediately above the focal plane. A particle in focus at the top of theoptical disector therefore is seen in the lookup section and notcounted. A particle in focus at the bottom of the optical disector,which is in the reference section and therefore not in the lookupsection, is counted. Counting frame rules are applied when the particlefirst comes into focus. The total volume of the sample then can becalculated by determining the maximum pellet size, section thickness,total sections per sample, sample volume and the paraffin mold base.Additionally, computerized image processing utilizing filters (such as,but not limited to, area, aspect, perimeter, and radius ratio) appliedon the extracted data and counts allow for determination of cells/volumeof each section.

Example 3.2 Cell Viability after Cryopreservation

The viability of cells collected from the finished osteoconductivematrices is analyzed after cryopreservation and post-thaw. According tosome embodiments, at least 70% of the viable cells that were present onthe sample (i.e., rinsed osteoconductive matrix particles) prior tocryopreservation are present on the osteoconductive matrix. Theviability of these cells can be determined using commercially availablemethods, including but not limited to, for example, metabolic assays,such as involving luciferase, tetrazolium salts, for e.g.,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11) and dye exclusion assays such as Tryptan Blue.

Example 3.3 BMP-2 Content of Demineralized Cortical Bone

Studies to determine the stability of DCB supplemented with BMP-2 areperformed to confirm retention of osteoinductive potential aftercryopreservation. Briefly, osteoinductivity of DCB supplemented withBMP-2 is measured utilizing an ELISA assay. These measured amounts ofBMP-2 are compared to an in vitro animal model utilizing an athymicmouse where certain levels of BMP-2 are known to correlate to boneformation.

FIG. 1 shows a plot of BMP-2 (pg/g DCB) versus time (weeks). The levelsof BMP-2 remain about or above 10,000 pg/g DCB after 12 weeks post-thaw.

Example 3.4 Immunogenicity

The orthopedic implant is analyzed for immunogenicity.

Mixed Lymphocyte Reaction (MLR)

Generally, the MLR is carried out by co-incubating lymphocytes from twostrains that differ in histocompatibility genes for several days.³H-thymidine then is added; the extent of its incorporation into DNAmeasures the proliferative response of T cells of one lymphocytepopulation to histocompatibility antigens of the other.

In order to examine the immunogencity of the orthopedic implant, up to14 samples were analyzed for their stimulatory effects in the MLR. Thesamples were tested against blood cells from three normal human donors.

To this end, cryopreserved tissue samples were thawed in a 37° C. waterbath until crystals were gone (about 20 min). Cryoprotectant solutionthen was removed; warm 5% dextrose in lactated ringers solution wasadded to the tissue samples and incubated for 5 minutes. Mitomycin C (30μg/ml) in culture medium (RPMI (Roswell Park Memorial Institute)supplemented with 2 mM L-glutamine and penicillin (10/mL)) then wasadded to the samples and incubated at 37° C. for 30 minutes. Testsamples were treated with Mitomycin C so that they would not give asignal alone. The samples were cut into small pieces, washed four timesin culture medium, and similar amounts of culture medium were added towells.

Cell samples were warmed in a 37° C. water bath. Following incubation,the cell samples were centrifuged at 1000 rpm for 5 min and thesupernatant (cryoprotectant solution) was removed. The pelleted cellswere resuspended in culture medium containing 30 μg/mL of Mitomycin-Cand incubated for 30 min at 37° C. The cell samples were centrifugedagain at 1000 rpm for 5 min, washed four times with RPMI medium, andresuspended in 4 mL of RPMI. For testing, cell numbers were adjusted to1×10⁶/mL.

For MLR assay, 30-50 mL blood in a heparinized syringe from each ofthree healthy volunteers was obtained. Two aliquots of D-PBS(phosphate-buffered saline) in sterile 50 ml screw cap and three tubesof Ficoll-Hupaque were prepared. 25 mL of blood was added to the sameamount of PBS in two tubes per donor, and 15 mL of blood was layered to33 mL of Ficoll-Hypaque. The tubes then were centrifuged atapproximately 1400 rpm for 30 min at room temperature, and 10 mL ofculture medium was aliquoted to three tubes. The plasma/platelet layerwas discarded from each Ficoll-Hypaque gradient, and the cells at theinterface from each of the tree tubes per donor were collected. 10 mL ofculture medium then was added to the tubes and centrifuged at 1400 rpmfor 15 min at room temperature. The red blood cell layer was treatedwith dispatch or bleach and discarded. The supernatant from each tubewas decanted and the cell pellets were resuspended in approximately 10mL of culture medium (combining three tubes into one). The tubes werecentrifuged at approximately 1200 rpm for 10 min at room temperature.The pelleted cells were resuspended in RPMI-10 and cell numbers wereadjusted to 1×10⁶ cells/ml. 3 mL of the cell suspension was kept andtreated with 30 μg/mL Mitomycin-C. The prepared cells then were added to0.1 ml of cells/well or to 0.1 ml of sample/well for MRL assay. Fortissue samples, 0.1 mL of RPMI-10 medium was added to each tissue sampleand the tissue sample (without liquid) was added to cells.

After five days treatment, cells were labeled overnight withapproximately 0.5 μCi/well [³H] thymidine, harvested, and counted in ascintillation counter. All conditions were performed in replicate.Results were reported as incorporation of [³H] thymidine as a measure oflymphocyte proliferation. Proliferation was expressed as a StimulationIndex (SI), where SI=cpm (blood cells+test sample)/[cpm (blood cellsalone)+cpm (test sample alone)], which is what is represented on theY-axis in FIG. 2. Positive controls were normal blood donor cells mixedwith mitomycin-treated different normal blood cell donor cells todemonstrate a strong MLR. Negative controls were normal donor cellsalone and test samples alone (sample (11)-(14)).

TABLE 12 MLR assay results Stimulation Index Sample Donor A Donor BDonor C (1) #023-93615: Cancellous tissue w/out DCB (cryopreservedproduct) 0.9 1.1 0.4 (2) #005-93647: Cancellous tissue pre-washed sample(cryopreserved) 0.7 0.9 0.2 (3) #005-93647: Cancellous tissue w/out DCB(Cryopreserved product) 0.6 0.8 0.3 (4) #005-93647: Cancellous tissuew/DCB (cryopreserved product) 0.5 1.0 0.3 (5) #005-93647: Cancelloustissue w/DCB another sample 0.5 1.1 0.3 (cryopreserved product) (6)#023-93615: Heat-inactivated Cancellous tissue with DCB 0.4 1.0 0.4 (7)#023-93615: Digested Cancellous tissue (sent refrigerated) 0.3 0.6 0.3(8) #005-93647: Digested Tissue (sent refrigerated) 0.3 0.6 0.5 (9)#023-93615: Digested cells from Cancellous tissue (sent 1.6 0.8 0.7refrigerated) (10) #005-93647: Digested Cells (sent refrigerated) 0.60.5 0.1 (11) Control: Trinity ® 1cc Same Sample as 11 (normal thaw,vial 1) 0.2 0.3 0.2 (12) Control Trinity ® 1cc Same Sample as 10 (thaw,centrifuged, 0.2 0.4 0.1 add cells to MLR with tissue for testing, vial2) (13) Control DCB 0.4 0.3 0.3 (14) Control DCB (second sample) 0.4 0.40.4 Positive Control (PC) Cells from Donor with Cells from a Different4.4 2.5 4.1 Donor treated with Mitomycin C

As shown in Table 12, none of the samples significantly stimulated a MLRas measured by the Stimulation Index when compared to the positivecontrols across blood donors. Some of the tissue/solid samples had arather high content of ³H-thymidine alone that may be due to passivetrapping.

FIG. 2 shows a plot of the MLR response of a negative control, DCB, and3 sample orthopedic implants prepared as described herein. The DCB, 3sample orthopedic implants, and negative control each demonstrated alower MLR response than the positive control.

Complement Activation: c3a Protein

The complement system is a complex system of proteolytic enzymes,regulatory and inflammatory proteins and peptides, cells surfacereceptors, and proteins capable of causing the lysis of cells (see, forexample, Fundamentals Immunology, 4th Ed., 1999. Paul, W. E. (Eds.),Lippincott-Raven Publishers, New York, N.Y.). The system can be thoughtof as consisting of three arrays of proteins. Two of these sets ofproteins, when engaged, lead to the activation of the third component ofcomplement (C3). The activation of C3 releases proteins that arecritical for opsonization (preparation for phagocytosis) of bacteria andother particles and engages the third set of proteins that insert intobiologic membranes and produce cell death through osmotic lysis. Inaddition, fragments generated from some of the complement components(for example, C3a and C5a) have potent inflammatory activities.

The two activation systems for C3 are referred to as the classicalpathway and alternative pathway. The classical pathway is initiated bythe formation of complexes of antigen with IgM and IgG antibody. Thisleads to the binding of the first component of complement, C1, and itsactivation, creating the C1 esterase that can cleave the next twocomponents of the complement system, C4 and C2.

C4 is a trimeric molecule, consisting of α, β, and γ chains. C1 esterasecleaves the α chain, releasing the C4b, which binds to surfaces in theimmediate vicinity of the antigen/antibody/C1 esterase complex. A singleC1 esterase molecule will cause the deposition of multiple C4bmolecules.

C2 is a single polypeptide chain that binds to C4b and is thenproteolytically cleaved by C1 esterase, releasing C2b. The resultingcomplex of the residual portion of C2 (C2a) with C4b (C4b2a) is a serineprotease whose substrate is C3. Cleavage of C3 by C4b2a (also referredto as the classical pathway C3 convertase) results in the release of C3aand C3b. The amplification nature of this system is implicit in thecapacity of a single antigen/antibody complex and its associated C1esterase to produce a large number of C3 convertases (i.e., C4b2acomplexes) and thus to cleave a large number of C3 molecules.

The components of the classical pathway can be activated by a distinct,non-antibody-dependent mechanism. The mannose-binding lectin (MBLectin)is activated by binding to (and being crosslinked by) repetitive sugarresidues such as N-acetylglucosamine or mannose. The activation ofMBLectin recruits the MBL-associated serine proteases MASP-1 and MASP-2,which are homologues of two of the constituent chains of C1 (C1r andC1s). This results in the activation of C4 and C2 and the formation ofthe classical pathway C3 convertase. Because the capsules of severalpathogenic microbes can be bound by MBLectin, this provides anantibody-independent pathway through which the complement system can beactivated by foreign microorganisms.

The alternative pathway can be activated by a variety of agents such asinsoluble yeast cell wall preparations and bacterial lipopolysaccharide.Antigen/antibody complexes also can activate the alternative pathway.The C3 convertase of the alternative pathway consists of a complex ofC3b (itself a product of cleavage of C3) bound to the b fragment of themolecule factor B. C3bBb is produced by the action of the hydrolyticenzyme, factor D, that cleaves the factor B; this cleavage only occurswhen factor B has been bound by C3b.

The alternative pathway also can act to amplify the classical pathwaybecause the C3 convertase of the classical system (C4b2a) provides asource of C3b that can strikingly enhance formation of the alternativepathway convertase (C3bBb) in the presence of factor D.

C3b, formed from C3 by the action of the C3 convertases, possesses aninternal thioester bond that can by cleaved to form a free sulfhydrylgroup. The latter can form a covalent bond with a variety of surfacestructures. C3b is recognized by receptors on various types of cells,including macrophages and B cells. The binding of C3b to antibody-coatedbacteria is often an essential step for the phagocytosis of thesemicrobes by macrophages.

C3b also is essential to the engagement of the terminal components ofthe complement system (C5 through C9) to form the membrane attackcomplex that causes cellular lysis. This process is initiated by thecleavage of C5. The C5 convertases that catalyze this reaction areC4b2a3b (the classical pathway C5 convertase) or a complex of C3bBb witha protein designated properdin (the alternative pathway C5 convertase).Cleaved C5, C5b, forms a complex with C6 and then C7, C8, and C9. ThisC5b/C9 complex behaves as an integral membrane protein that isresponsible for the formation of complement-induced lesions in cellmembranes. Such lesions have a donut-like appearance, with C9 moleculesforming the ring of the donut.

In addition to the role of the complement system in opsonization and incell lysis, several of the fragments of complement components formedduring activation are potent mediators of inflammation. C3a, a fragmentreleased by the action of C3 convertases, binds to receptors on mastcells and basophils, resulting in the release of histamine and othermediators of anaphylaxis. C3a and C5a (a fragment released by the actionof C5 convertases) are termed an anaphylotoxins. C5a also is achemoattractant for neutrophils and monocytes.

The process of activation of the complement cascade is highly regulated.Several regulatory proteins (for example, C1 esterase inhibitor, decayaccelerator factor, membrane cofactor protein) exist that function toprevent uncontrolled complement activation.

Complement activation from two proteins, C3a and SC5b can be assayed.Commercially available assays are available to measure C3a and SC5b,such as, but not limited to, BD OptEIA™ Human C3a ELISA (BD Biosciences)and MicroVue SC5b 9 Plus Enzyme Immunoassay (Quidel, San Diego, Calif.).Briefly, the BD OptEIA™ Human C3a ELISA is a solid phase sandwich ELISAthat utilizes a monoclonal antibody specific for human C3a-desArg coatedon a 96-well plate. Standards and samples are added to the wells, andany C3a-desArg present binds to the immobilized antibody. The wells arewashed and a mixture of biotinylated polyclonal anti-human C3a antibodyand streptavidin-horseradish peroxidase is added, producing anantibody-antigen-antibody sandwich. The wells are again washed and asubstrate solution is added, which produces a blue color in directproportion to the amount of C3a-desArg present in the initial sample.The Stop Solution changes the color from blue to yellow, and the wellsare read at 450 nm. Commercially available assays also are available tomeasure SC5b9 such as, but not limited to, MicroVue SC5b 9 Plus EnzymeImmunoassay (Quidel, San Diego, Calif.). Briefly, the assay measures theamount of the SC5b 9 complex present in human plasma or serum specimens.The Terminal Complement Complex (TCC, SC5b-9) is generated by theassembly of C5 through C9 as a consequence of activation of thecomplement system by either the classical, lectin or alternativepathway. The membrane attack complex (MAC), a form of TCC, is a stablecomplex that mediates the irreversible target cell membrane damageassociated with complement activation. Complexes formed in the absenceof a target membrane bind to naturally occurring regulatory serumproteins, e.g. the S protein, 5-7 at the C5b 7 stage of assemblyforming, soluble, non-lytic TCC. The assay measures the concentration ofTCC thereby giving an indication of the status of the terminalcomplement pathway in the specimen. It uses a monoclonal antibody to theC9 ring of TCC to capture the complex. The trapped TCC is subsequentlydetected with HRP-conjugated antibodies that bind to antigens of theSC5b 9 complex.

FIG. 3A shows a plot of the complement activation response of c3aprotein of a positive control, negative control, untreated plasma, and 4sample orthopedic implants. FIG. 3B shows a plot of the complementactivation response of SC5b protein of a positive control, negativecontrol, untreated plasma, and 4 sample orthopedic implants. The plotsindicate that the implants provoke no response in either the c3a (FIG.3A) or SC5b (FIG. 3b ) assay.

Example 3.5 Biocompatibility Testing (ISO10993)

The ISO10993 criteria are a series of standards for evaluating thebiocompatibility of a medical device. Table 13 shows several of thesetests and conclusions drawn therefrom for orthopedic implants preparedfrom a bone tissue.

TABLE 13 Biocompatibility Testing (ISO10993) Test ConclusionCytotoxicity, ISO Agar diffusion (n = 4) Not cytotoxic Acute SystemicToxicity in Mouse, systemic No systemic injection (n = 3) toxicity Acuteintracutaneous injection (n = 2) No irritation Rabbit pyrogen test(material mediated) (n = 2) Not pyrogenic Genotoxicity: AMES reversemutation study (n = 2) Not mutagenic Hemolysis-Human Blood, directcontact (n = 1) Not hemolytic Hemolysis-Human Blood, indirect contact (n= 2) Not hemolytic

Example 3.6 Rat Intramuscular Study

The three components of the orthopedic implant (osteoinductivecomponent, osteoconductive component, and osteogenic cell component)from a bone tissue were measured at 28 days (the end point for atopicbone formation in a muscle pouch) in vivo using a well-establishedathymic rat muscle pouch model. Briefly, two test groups wereestablished: (1) N₁=8 per test group, randomized, bilateralimplantation, and (2) N₂=10 sections/implant. The best of 5 slides werescored for each test group on a grade of 0-4. Table 14 shows resultsfrom the intramuscular study where scores greater than or equal to 1.0pass the U.S. Food and Drug Administration osteoinductive test.

TABLE 14 Athymic rat muscle pouch model assessment of differentiationSample Score Day 14 IM orthopedic implant 1 1.25 orthopedic implant 1with freeze/thaw 0.86 orthopedic implant 2 1.86 Day 28 IM orthopedicimplant 1 1.65 orthopedic implant 1 with freeze/thaw 0.88 orthopedicimplant 2 1.57

FIG. 4A shows that at day 14, hypotrophic chondrocytes are beginning toform; FIG. 4B shows that at day 28, new bone formation is evident.

Example 3.7 Rat Posterolateral Spinal Fusion

Orthopedic implants from a bone tissue were implanted into a spinalfusion model. An incision of approximately 4 cm is made on the posteriormidline over the distal lumbar spine. The transverse processes of the L4and L5 are exposed and decorticated to allow blood flow. The bone graftis then placed over the decorticated surfaces in the space betweenposterolateral vertebral processes of an athymic rat. If the implant isosteogenic, bone will grow and the spine will fuse. The spine is excisedand rigidity of the spine tested 8 weeks post-implantation utilizing themanual bend test.

TABLE 15 Results at 8 weeks post-implantation by the manual bend test:Sample Fusion orthopedic implant 1 (30% DCB) 2/7 (29%) orthopedicimplant 1 with freeze/thaw 2/8 (25%) orthopedic implant 2 1/6 (17%)

Table 15 shows results obtained at 8 weeks post-implantation by themanual bend test. Less subjective radiographic data was obtained by CT.Radiographs were scored based on a 6-point grading system: 1) 0 points(no bone formation); 2) 1 point (bone filling less than 25% of area); 3)2 points (bone filling 25-50% of area; 4) 3 points (bone filling 50-75%of area); 5) 4 points (bone filling 75-99% of area); and 6) 5 points(clear evidence of fusion with the bone filling all gaps between L4 andL5).

Table 16 shows that some explants scored a grade 5 (fusion) byradiographic analysis that were not considered fused using the manualbend test. It was concluded that fusion had occurred because of theconductive effect of the matrix and because the inductive effect of BMPwas still present. Radiographic results were graded on a scale of 0-5,with N=3 observers (Grade ≧4 (75-99%; 100%): Fusion in the manual bendtest is equivalent to a grade 5 radiograph score.

TABLE 16 Results obtained Post-implantation Radiographically by CTRadiographic Sample Grade orthopedic implant 1 (30% DCB) 5.7/7 (81%)orthopedic implant 1 with freeze/thaw 4.7/8 (59%) orthopedic implant 24.7/6 (78%)

Because there is a 20% increase in fusion when cells are present, theseresults suggest that the osteogenic cells together with theosteoinductive components affect bone formation. The presence ofosteoconductive, osteoinductive and osteogenic components in theimplants of the described invention provided bone formation earlier intime, when compared to controls.

Example 4 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Acetic Acid Rinse

One left ilium from Donor 007-63609 was processed to obtain freshcancellous bone particles.

Cutting the Tissue

The bulk of the soft tissue was removed from the ilium. The acetabulumwas removed and release notches were cut along the iliac crest. Theiliac crest was then removed with cutting following its contour. Thearea of the iliac spine was cut until there was no more growth platevisible via cross section. Any areas of heavy cortical bone and anyremaining portions of periosteum were trimmed off. The ilium was thencut into approximately 3 cm by 3 cm cubes.

Milling and Rinsing of Tissue

Prior to placing tissue in the mill, all grinding parts of the mill werewet down with 4° C. phosphate buffered saline solution (PBS). The tissuewas placed into the mill. Ground cancellous bone was generated, sized <2mm in diameter. The tissue was then transferred to a room temperatureNalgene container. The tissue was subjected to a series of rinses withcold PBS, followed by a rinse in acetic acid solution for 5 minutes andthen followed again by a series of rinses with cold PBS.

Between each rinse, the rinsing solution was decanted off using a sieveto catch the tissue. After the acetic acid rinse, a new Nalgenecontainer as well as a new sieve were used in order to minimize thetissue's exposure to residual acetic acid. After the last rinsing step,the tissue was then prepared for cryopreservation. A sample was setaside in order to evaluate the cell metabolic activity of the tissueusing commercially available methods, including but not limited to, forexample, metabolic assays, such as involving luciferase, tetrazoliumsalts, for e.g.,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11) and dye exclusion assays such as Tryptan Blue.The assay confirmed that the tissue contained metabolically active cellswithin its matrix.

Cryopreservation of Tissue

Mesencult basal media was prepared and sterile filtered. Cryoprotectant(10 ml) was added in order to assure full coverage of the tissue. Thesamples were packaged in one layer of Teflon pouches (CryoSystems). Onesample as a probe sample was cryopreserved in a laboratorycryopreservation unit. Once the program cycle was complete, the tissuewas placed in liquid nitrogen.

It should be understood by those skilled in the art that manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to improve, forexample, but not limited to, efficiency, and yield.

Plating of Tissue for Mesenchymal Stem Cell Characterization (Donor035-59660)

After the last rinsing step, two 6-well tissue culture plates wereseeded with approximately 1-2 cc of tissue per well in 4 ml of MSCcomplete media (Mesencult Basal Media with MSC supplements and 1×pen/strep). The media were changed on the tissue every 2-3 days asneeded using aseptic cell culture techniques. The tissue was removedcarefully from the culture using sterile forceps after approximately twoweeks. While the larger sized pellets were removed, some very smallpieces remained on the plate. After two weeks of incubation severalcells appeared in each well. The cells were allowed to continue to growfor use in various cell characterization assays described below. Thecultured cells were able to differentiate into the osteogenic andadipogenic lineages as demonstrated by the presence of alkalinephosphatase, mineralization of the cell layer, and the presence of lipidcontaining vesicles within the cells.

Osteogenic Differentiation Assay: Alkaline phosphatase & Von KossaStaining

After cell proliferation was observed, the cells were transferred to 25ml t-flasks. After adherence of the MSC's after sub-culturing, the cellswere exposed to osteogenic complete medium (Stem Cell Technologies).After five days of incubation, the old medium and non-adherent cellswere removed and the medium replaced with fresh complete medium. Themedia were exchanged every 2-3 days as needed using aseptic cell culturetechniques for 6 weeks. Once the multi-layering of the cells wasobserved, 13-Glycerophosphate was added to the medium. After 6 weeks inculture the cells were fixed and stained using alkaline phosphatase andvon kossa.

Adipogenic Differentiation Assay: Oil Red O Staining

After subculturing and adherence of the MSC's, the cells were exposed toadipogenic complete medium (Stem Cell Technologies). The media wasexchanged every 2-3 days as needed using aseptic cell culture techniquesfor 3 weeks. As the cells divided and started forming a multi-layerstate, the formation of fat vacuoles was observed. After 3 weeks inculture the cells were fixed and stained using Oil Red 0.

Flow Cytometry of Tissue Derived MSC's, Donors 035-59660 & Donor002-57470:

The cells from Donor 035-59660 and Donor 002-57470 used for thedifferentiation assays were expanded and used for flow cytometryanalysis. The cells were compared to an established MSC line (humanMesenchymal Stem Cells, Catalog No. MSC-001F, Stem Cell Technologies,Vancouver, BC, CN) as well as to KG1A cells (which served as a negativecontrol for MSC markers and a positive control for hematopoietic markersCD34 and CD45). Isotype controls were measured as well as unstained,single and triple stain controls. The isotype controls are used toconfirm that the primary antibody binding is specific and not due toother protein interactions or non-specific binding. The stain controlsare used in order to determine background autofluorescence of the cells,as well as the spectral overlap of the fluorochromes in the cytometeralignment.

The cells were reacted with CD 34, 44, 45, 90, 105 and 166 antibodies.MSC's were expected to be positive for CD44, 90, 105 and 166 andnegative for CD34 and 45. The KG1a cells were expected to be positivefor CD34 and 45 and were used as negative control markers for the MSC's.The following antibody and conjugate pairs were used:

TABLE 17 Flow Cytometry Cell Surface Marker Antibodies Antibody andVolume to use conjugate per 500,000 cells CD34 APC 10 μL CD44 FITC 20 μLCD166 PE 20 μL CD45 APC 10 μL CD90 FITC 20 μL CD105 PE 20 μL IgG1 APC 10μL IgG1 FITC 20 μL IgG1 PE 20 μL IgG3 PE  5 μL

The following experimental setup was used to characterize cells by flowcytometry:

TABLE 18 Experimental setup used to characterize cells by flow cytometrySingle Stain CD 34 CD 44 CD 166 CD 45 CD 90 CD 105 compensation (500 μl(500 μL (500 μL (500 μl (500 μL (500 μL controls KG-1a) MSCs) MSCs)KG-1a) MSCs) MSCs) Triple Stain CD 34 CD 45 Compensation CD 44 CD 90Controls CD 166 CD 105 (250 μl (250 μl KG-1a KG-1a and and 250 μL 250 μLMSCs) MSCs) Isotype IgG1 IgG1 IgG1 IgG1 Controls APC APC APC APC IgG1IgG1 IgG1 IgG1 Fitc Fitc Fitc Fitc IgG1 PE IgG3 PE IgG1 PE IgG3 PE (500μL (500 μL (500 μl (500 μl) MSCs) MSCs) KG- 1a) KG-la Unstained KG-1a500 μL Controls 500 μL MSC line Test Articles 035- 002- 035- 002- 5966057470 59660 57470 CD 34 CD 34 CD 45 CD 45 CD 44 CD 44 CD 90 CD 90 CD 166CD 166 CD 105 CD 105 Test Article 035- 035- 035- 035- Isotype 5966059660 59660 59660 controls IgG1 IgG1 IgG1 IgG1 APC APC APC APC IgG1 IgG1IgG1 IgG1 Fitc Fitc Fitc Fitc IgG1 PE IgG3 PE IgG1 PE IgG3 PE 002- 002-002- 002- 57470 57470 57470 57470 IgG1 IgG1 IgG1 IgG1 APC APC APC APCIgG1 IgG1 IgG1 IgG1 Fitc Fitc Fitc Fitc IgG1 PE IgG3 PE IgG1 PE IgG3 PE

As shown in Table 19, flow cytometry analysis confirmed that theosteogenic cells obtained from the fresh cancellous bone were positivefor CD44, 90, 105 and 166 and negative for CD34 and 45, indicating thatthe osteogenic cells comprise MSC's. In contrast, the KG1a cells werepositive for CD34 and 45 and negative for CD44, 90, 105 and 166. Thefollowing percentages were representative of the surface markers for theMSC cell line, tissue derived MSC's and the KG1A cells:

TABLE 19 Flow Cytometry Results Obtained for Osteoconductive Matricesobtained from Two Donors Tissue derived Tissue derived from MSC's fromMSC's MSC from MTF035- from MTF002- KG1a cell line 59660 [Donor 1] 57470[Donor 2] cell line CD44 94.07% 89.69% 83.11%  0.20% CD90 92.84% 92.55%80.95%  0.24% CD105 98.27% 92.82% 82.07%  0.05% CD166 97.06% 89.56%82.96%  0.66% CD34  0.62%  1.99%  1.12% 98.54% CD45  0.19%  1.46%  3.09%98.82%

Example 5 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with NH₄Cl Rinse

One left ilium was processed from Donor 041-59941 to obtain cancellousbone particles to compare the rinsing process with acetic acid and therinsing process with ammonium chloride.

Cutting the Tissue

The bulk of the soft tissue was removed from the ilium. The acetabulumwas removed and release notches were cut along the iliac crest. Theiliac crest was then removed with cutting following its contour. Thearea of the iliac spine was cut until there was no more growth platevisible via cross section. Any areas of heavy cortical bone and anyremaining portions of periosteum were trimmed off using the band saw.The ilium was then cut into approximately 3 cm by 3 cm cubes.

Milling & Rinsing of Tissue

Prior to placing tissue in the mill, all grinding parts of the mill werewet down with 4° C. phosphate buffered saline solution (PBS). The tissuewas placed into the mill. Ground cancellous bone was generated, sized <2mm in diameter. The tissue was then divided in half and transferred totwo room temperature Nalgene containers. The tissue was subjected to aseries of rinses with cold PBS, followed by a rinse in acetic acidsolution for 5 minutes and then followed again by a series of rinseswith cold PBS.

Between each rinse, the rinsing solution was decanted off using a sieveto catch the tissue. After the acetic acid rinse, a new Nalgenecontainer as well as a new sieve was used in order to minimize thetissue's exposure to residual acetic acid.

Between each rinse, the rinsing solution was decanted off using a sieveto catch the tissue. After the ammonium chloride rinse, a new Nalgenecontainer as well a new sieve was used in order to minimize the tissue'sexposure to residual ammonium chloride.

At the end of the rinsing steps, a sample was set aside from eachrinsing process in order to evaluate the cell metabolic activity of thetissue via methyl tetrazolium (MTS) assay.

The assay confirmed that the metabolic activity of the cells after theacetic acid rinse was unchanged. The assay for the ammonium chlorideshowed interaction between the chemical and the assay which made itunable to give us accurate metabolic results. Through visual inspectionof the tissue, the tissue rinsed using acetic acid appeared to containless blood products than the tissue rinsed using the ammonium chloride.

Example 6 Fabrication of Demineralized Cortical Bone Particles

Human long bone is recovered aseptically from a deceased donor under theage of 65 years, and stored at 4° C. until ready for processing. Thebone is debrided and the shaft of the bone is cut into bulk pieces. Aninitial cleaning is performed during which the pieces of bone are soakedin a surfactant solution, and then soaked in a disinfectant solution,followed by a water rinse. The bone pieces are then allowed to soak inethanol, and dried.

To create demineralized bone particles, the bone sections are milled topowder and sieved to capture particles having sizes in the range ofabout 212 μm to about 850 μm. The particles are cleaned with hydrogenperoxide solution, then rinsed in water, and soaked in about 70%ethanol, and finally air dried in a sieve connected to a vacuum.

The air-dried bone particles are demineralized by agitating them in 0.6N HCl, followed by one or more soaks in fresh 0.6 N HCl for a sufficientperiod of time to remove the endogenous calcium salts. The boneparticles are then rinsed in water, soaked in 0.1 M sodium phosphatedibasic, then rinsed in water several more times. The bone particles arethen air dried in a sieve connected to a vacuum to a residual moisturecontent in the range of about 15-25%.

Example 7 Fabrication of Demineralized Cortical Bone Fibers

Human long bone is recovered aseptically from a deceased donor under theage of 65 years and stored at 4° C. until ready for processing. The boneis debrided and the shaft of the bone is cut into cross-sections. Aninitial cleaning is performed during which the cross-sections of boneare soaked in a surfactant solution, and then soaked in a disinfectantsolution, followed by a water rinse. The bone sections are then allowedto soak in ethanol, and dried.

To create demineralized bone fibers, the bone sections are first shavedacross the shaft using a controlled advancement rate of a lathe bithaving a width approximately equal to the desired length of the bonefibers. The bone fibers are demineralized by agitating them in 0.6 N HClfor a sufficient period of time to remove the endogenous calcium salts,after which the fibers are agitated, successively, with water, 0.1 Msodium phosphate dibasic, water again, then PBS. The bone fibers arethen air dried in a sieve connected to a vacuum to a residual moisturecontent in the range of about 60-80%.

Example 8 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Demineralized Cortical Bone Particles Added

Human ilium is processed from a donor to obtain cancellous boneparticles, prepared as in Example 4, to which demineralized corticalbone (DCB) particles, prepared as in Example 6, are added. Thefreshly-milled cancellous bone particles are mixed with the DCBparticles in a ratio of about one-third DCB particles to abouttwo-thirds cancellous bone particles (w/w) in such a manner as to ensurea homogenous mixture.

The mixture of DCB particles and cancellous bone particles is scoopedinto a suitable container, which is then filled with a basal cellculture media containing 10% dimethyl sulfoxide (DMSO) as acryoprotectant to maintain the viability of the cells in the cancellousbone particles during freezing and thawing. The mixture is agitated tosuspend the DCB particles and cancellous bone particles in thecryoprotective media, and then frozen at a controlled rate to about−100° C. and stored in liquid nitrogen vapor phase at −185° C. forlong-term storage.

Example 9 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Demineralized Cortical Bone Fibers Added

Human ilium is processed from a donor to obtain cancellous boneparticles, prepared as in Example 4, to which demineralized corticalbone (DCB) fibers, prepared as in Example 7, are added. The freshlymilled cancellous bone particles are mixed with the DCB fibers in aratio of about 60% DCB fibers to about 40% cancellous bone particles(w/w) in such a manner as to ensure a homogenous mixture.

The mixture of DCB fibers and cancellous bone particles is scooped intoa suitable container, which is then filled with a basal cell culturemedia containing 10% dimethyl sulfoxide (DMSO) as a cryoprotectant tomaintain the viability of the cells in the cancellous bone particlesduring freezing and thawing. The mixture is agitated to suspend the DCBfibers and cancellous bone particles in the cryoprotective media, andthen frozen at a controlled rate to about −100° C. and stored in liquidnitrogen vapor phase at −185° C. for long-term storage.

Example 10 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Crushed Demineralized Cancellous Bone Particles Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which crushed demineralized cancellous bonematrix is added.

Example 11 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Autologous Bone Marrow or Other Autologous Cells Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which autologous bone marrow or otherautologous cells is added. Generally, autologous bone marrow or otherautologous cells can be added to the cancellous bone particles obtainedas in Example 4 to increase the amount or volume of the orthopedicimplant thereby increasing the possibility of fusion.

Example 12 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Growth Factor or Factors Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which at least one growth factor is added.

Example 13 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Growth Factors and Demineralized Bone Matrix Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which at least one growth factor anddemineralized bone matrix are added.

Example 14 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Growth Factors and DCB Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which at least one growth factor and DCBare added.

Example 15 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Growth Factors and Crushed Cancellous Bone Matrix Added

Human ilium is processed from a Donor to obtain cancellous particles asin Example 4 to which at least one growth factor and crushed cancellousbone matrix are added.

Example 16 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Growth Factors and Autologous Bone Marrow Cells or OtherAutologous Cells Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which at least one growth factor andautologous bone marrow cells or other autologous cells are added.Generally, autologous bone marrow or other autologous cells can be addedto the cancellous bone particles obtained as in Example 4 to increasethe amount or volume of the orthopedic implant thereby increasing thepossibility of fusion.

Example 17 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Demineralized Bone Matrix and Autologous Bone Marrow Cells orOther Autologous Cells Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which demineralized bone matrix andautologous bone marrow cells or other autologous cells was added.Generally, autologous bone marrow or other autologous cells can be addedto the cancellous bone particles obtained as in Example 4 to increasethe amount or volume of the orthopedic implant thereby increasing thepossibility of fusion.

Example 18 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Crushed Demineralized Cancellous Bone Matrix and AutologousBone Marrow Cells or Other Autologous Cells Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which crushed demineralized autologouscortical bone and autologous bone marrow cells or other autologous cellswas added. Generally, autologous bone marrow or other autologous cellscan be added to the cancellous bone particles obtained as in Example 4to increase the amount or volume of the orthopedic implant therebyincreasing the possibility of fusion.

Example 19 Fabrication of Fresh Cancellous Bone Particles from HumanIlium with Biological Components Added

Human ilium is processed from a Donor to obtain cancellous boneparticles as in Example 4 to which biological components was added.Biological components include, but are not limited to, DNA, RNA, shorthairpin RNA (shRNA), small interfering RNA (siRNA), micro RNA (miRNA),polysaccharides, peptides, matrix proteins, glycosaminoglycans such as,but not limited to, hyaluronic acid, viral vectors, and liposomes.

Example 20 Size Characterization of Demineralized Bone Fibers

A solution of surfactant in deionized ultrafiltered water is added to aportion of a sample of dehydrated demineralized bone fibers andultrasonically agitated to hydrate and disperse the bone fibers. A dyeis added to the mixture (e.g., a fluorescamine dye solution) and, setfor a sufficient time to allow the dye to react with the bone fibers.The bone fibers are then dispersed in the mixture, and collected on apolycarbonate filter membrane by vacuum filtration. Multiple portions ofthe sample may be collected for analysis as described below.

To determine the lengths and widths of the collected bone fibers, eachfilter membrane is placed under lighting conditions appropriate for thedye used (e.g., under UV light for fluorescamine dye), and each quadrantof the filter is separately imaged for visual examination. A length andwidth chord is indicated on the image of each fiber as estimates of thelength and width of the fiber. The indication and measurement of eachchord may be made by a trained technician or an algorithm usingcalibrated imaging software. Clumps of fibers are excluded from theanalysis. A statistical analysis of the measured lengths and widths isperformed to determine length and width characteristics of thepopulation of bone fibers in the samples.

Example 21 Thawing Osteogenic Implants

In some embodiments, prior to implantation, a cryopreserved orthopedicimplant with or without DCB is thawed. The DCB may be in the form ofparticles as in Example 6 or fibers as in Example 7. The thaw procedurewarms the tissue preparing it for surgical implantation. The vialcontaining cryopreservation solution and tissue is thawed in salinewarmed to 37° C. to expedite the thawing process. Once thecryopreservation solution is free flowing, the cryopreservation solutionis decanted from the vial. Then the tissue is rinsed with a washsolution well known in the art, including but not limited to saline, 5%dextrose in lactated ringers solution, phosphate buffered saline, andany additional isotonic solution. Prior to implantation, decant the washsolution. In some embodiments, DCB fibers may be mixed into theorthopedic implant after the orthopedic implant is thawed, mixing theimplant and DCB fibers so as to ensure a homogenous mixture.

The wash solution, prior to application to the tissue, is warmed to atemperature, not exceeding 35-39° C. in order to minimize any damage tothe cells contained in the tissue. The wash solution is exchangedthroughout the rinse. Any remaining tissue from the surgery is notre-frozen for future use. All remaining tissue is disposed ofappropriately after surgery.

A strainer is optionally used to contain the tissue during the decantingprocess. This allows the cryopreservation solution and rinsate to beremoved from the tissue while minimizing any possible contamination oftissue during preparation from human contact. Optionally, gauze also isused to contain the tissue during the decant/thaw procedure.

Example 22 Comparison of Frozen and Thawed Cancellous Bone OrthopedicImplant Comprising an Osteoconductive Matrix According to the DescribedInvention to a Commercially Available Frozen Orthopedic Implant(Commercial Matrix)

The impact of the thaw procedures for both orthopedic implants of thepresent invention and a commercially available frozen orthopedic implantare compared for loss of viable cells during the thaw and rinsingprocess prior to implantation. The cryopreservation solution from eachimplant is analyzed for the presence of viable cells using commerciallyavailable methods, including but not limited to, for example, metabolicassays, such as involving luciferase, tetrazolium salts, for e.g.,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11), and dye exclusion assays such as TryptanBlue.

Example 23 Comparison of Fresh Cancellous Bone Allograft with DCB of theDescribed Invention Versus Commercial Matrix: Rat Spinal Fusion Study

The following specimens were implanted into a spinal fusion model: a)donor 1 orthopedic implant; b) donor 2 orthopedic implant; c) Donorfreeze/thawed orthopedic implant; d) Commercial Matrix implant; and f)Freeze/Thaw Commercial Matrix implant. An incision of approximately 4 cmis made on the posterior midline over the distal lumbar spine. Thetransverse processes of the L4 and L5 are exposed and decorticated toallow blood flow. Each specimen then is placed over the decorticatedsurfaces in the space between posterolateral vertebral processes of anathymic rat.

At the 8 week end point, the spine was removed and fusion was determinedby a manual bend method. Using the manual bend test as a measure thefollowing fusion results were defined: 1) 2/7 fused from Donor 1; 2) 2/8fused from Freeze/Thaw Donor 1; 3) 1/6 fused from Donor 2; 4) 0/8 fusedfrom Commercial Matrix; and 5) 0/8 fused from Commercial MatrixFreeze/Thaw.

Radiographs also were taken at 2, 4, 6 and 8 weeks. Spines are selectedfor microCATscan. Histology is prepared for all samples after imaging iscomplete.

The radiographs were scored based on a 6-point grading system: 1) 0points (no bone formation); 2) 1 point (bone filling less than 25% ofarea); 3) 2 points (bone filling 25-50% of area; 4) 3 points (bonefilling 50-75% of area); 5) 4 points (bone filling 75-99% of area); and6) 5 points (clear evidence of fusion with the bone filling all gapsbetween L4 and L5).

Based on analysis of the 8 week results, the following percentradiographs scored a grade of 4 or better (75-100% bone filling ofarea): a) 81% Donor 1; b) 78% Donor 2; c) 59% Freeze/Thaw Donor 1; d) 9%Commercial Matrix; and e) 41% Freeze/Thaw Commercial Matrix.

II. Source Tissue Adipose Tissue Example 24 Fabrication of an Implant

Adipose tissue comprising an endogenous stem cell niche is recoveredaseptically from a cadaveric donor within 24 hours post mortem or from aliving donor undergoing elective liposuction surgery. For example,Visceral fat can be excised from cadaveric donors or obtained withconsent from living donors undergoing elective procedures, such asliposuction, from body regions rich in adipose, for example, hip, thighand abdomen. Subcutaneous adipose can be procured from the hypodermis bydissection from full thickness skin excised from a cadaveric donor.Adipose tissue from infrapatellar fat pads can be dissected out duringrecovery of knee-en-bloc from a cadaveric donor. The adipose tissue isstored at 4° C. until ready for processing. Generally, tissue processingcommences within 72 hours post-mortem. The adipose tissue is exposed toa bioburden reducer to generate preprocessed adipose tissue. Thepreprocessed adipose tissue is subjected to a series of PBS soaks withagitation. The preprocessed agitated adipose tissue then is minced andsubjected to a series of rinses with cold PBS, followed by a rinse inacetic acid solution and then followed again by a series of rinses withcold PBS. The pH of the rinseate is at or near physiological pH at theend of the rinses.

Additionally, after the last rinsing step, the tissue is prepared forcryopreservation.

Additionally, a sample is set aside in order to evaluate the metabolicactivity of the tissue using commercially available methods, includingbut not limited to, for example, metabolic assays, such as involvingluciferase, tetrazolium salts, for e.g.,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11) and dye exclusion assays such as Tryptan Blue.

Additionally, prior to cryopreservation, one or more growth-inductivecomponents are optionally added. Examples of the growth-inductivecomponents that can be added include, but are not limited to, BMP-2 and4, VEGF, bFGF, TGF-β, NELL-1, PDGF, and/or a combination thereof. Forcryopreservation, for example, Mesencult basal media is prepared andsterile filtered. Cryoprotectant (10 ml) is added in order to assurefull coverage of the tissue. Exemplary cryoprotectant include but arenot limited to dimethyl sulfoxide (DMSO), glycerol, ethylene glycol,propylene glycol, 2-Methyl-2.4-pentanediol (MPD), and sucrose. Thesamples are packaged in cryoresistant containers. The packaged tissuethen is subjected to slow controlled rate freezing to at least −80° C.and placed in liquid nitrogen.

It should be understood by those skilled in the art that manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to improve, forexample, but not limited to, efficiency, and yield.

Example 25 Fabrication of an Implant by Reseeding Adipose-Derived StemCells on a Decellularized Matrix

Adipose tissue with its endogenous stem cell niche is recoveredaseptically from a cadaveric donor within 24 hours post mortem or fromliving donors undergoing elective liposuction surgery. For example,visceral fat can be excised from cadaveric donors or obtained withconsent from living donors undergoing elective procedures, such asliposuction, from body regions rich in adipose, for example, hip, thighand abdomen. Subcutaneous adipose can be procured from the hypodermis bydissecting it out from full thickness skin excised from cadaveric donor.Adipose tissue from infrapatellar fat pads can be dissected out duringrecovery of knee-en-bloc from cadaveric donor. The adipose tissue isstored at 4° C. until ready for processing. Generally, tissue processingcommences within 72 hours post-mortem. The adipose tissue is exposed toa bioburden reducer to generate preprocessed adipose tissue. Thepreprocessed adipose tissue is subjected to a series of PBS soaks withagitation. The preprocessed agitated adipose tissue is then chopped intosmall pieces approximately 0.5×0.5×0.5 cm. The chopped adipose piecesare then subjected to a series of rinses with cold PBS. The pH of therinseate is at or near physiological pH at the end of the rinse. Therinseate is divided into two batches for stem cell isolation anddecellularized matrix preparation.

Isolation of ASCs

Viable Adipose-derived stem cells (ASCs) are isolated according toestablished protocols (Young et al., 2011, Acta Biomaterialia, 7:1040-1049). Briefly, following rinses with a buffered saline solution(e.g., 0.01M PBS, pH 7.4), one batch of the rinsed tissue is digestedwith a dissociation agent (e.g., collagenase) in order to disperse thetissue while maintaining cell viability. The digest is subjected tocentrifugation to separate the stromal vascular fraction (SVF) rich inadipose-derived stem cells from the supernatant rich in lipid filledadipocytes and matrix. The supernatant is aspirated and the aspirate isfrozen at −80° C. until further use. The SVF pellet is resuspended inPBS washing solution and is subjected to a series of cold PBS washeswith alternating steps of centrifugation. Following the final wash andresuspension in PBS, the resuspended solution is subjected to filtrationto remove undigested tissue and to obtain isolated SVF enriched withASCs for seeding. Alternatively, ASCs can be isolated from the othercells present in digested adipose tissue on the basis of cell size orimmunohistochemically, for example, by using magnetic beads, affinitychromatography, fluorescence-activated cell sorting (FACS), flowcytometry, or with a suitable device.

Additionally, the isolated ASCs express antigens, including, but notlimited to, CD73, CD90, CD29, CD44, CD105, and/or a combination thereof.Additionally or alternatively, the isolated ASCs do not expressantigens, including, but not limited to CD33, CD34, CD45, CD4, CD31,CD62p CD14, HLA-DR, and/or combination thereof.

Additionally, a sample is set aside in order to evaluate the biologicalactivity of the tissue using commercially available methods, including,but not limited to, for example, metabolic assays, such as involvingluciferase, tetrazolium salts, for e.g.,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11), and dye exclusion assays such as TryptanBlue.

Alternatively, isolated ASCs can be cultured without differentiationusing standard culture media typically supplemented with 5%-20% serum.For example, the ASCs are passaged at least 5 times in such mediumwithout differentiating, while still retaining their multiplicity.Adipose-derived stem cells can be maintained in control medium until 80%confluent. Cells are harvested at confluence and population doublingcalculated using the formula log N₁/log N₂, where N₁ is the number ofcells at confluence prior to passaging and N₂ is the number of cellsseeded after passaging. Cumulative population doubling is determined incultures maintained until passage 13 (approximately 165 days). The meancumulative population doubling obtained from 3 donors is expressed as afunction of passage number.

Confirmation of Multi-Lineage Differentiation of Adipose-Derived StemCells

Adipose-derived stem cells at passage 1 can be analyzed for theircapacity to differentiate toward the adipogenic, osteogenic,chondrogenic, and myogenic lineages. To induce differentiation, the stemcells are cultured with specific induction media as detailed in Table20.

TABLE 20 Lineage-specific differentiation induced by mediasupplementation Medium Media Serum Supplementation Control DMEM 10% FBSnone Adipogenic (AM) DMEM 10% FBS 0.5 mM isobutyl- methylxanthine(IBMX), 1 μM dexamethasone, 10 vM insulin, 200 μM indomethacin, 1%antibiotic/antimycotic Osteogenic (OM) DMEM 10% FBS 0.1 μMdexamethasone, 50 μM ascorbate-2- phosphat, 10 mM β- glycerophosphate,1% antibiotic/antimycotic Chondrogenic (CM) DMEM 1% FBS 6.25 μg/mlinsulin, 10 ng/ml TGFβ1, 50 nM ascorbate-2- phosphate, 1%antibiotic/antimycotic Myogenic (MM) DMEM 10% FBS, 0.1 μM 5% HSdexamethasone, 50 μM hydrocortisone, 1% antibiotic/antimycotic

Each media has been previously described and shown to inducemulti-lineage differentiation of MSCs (Pittenger, M. et al, 1999,Science 284: 143-147; Grigoradis, A. et al., 1988, J. Cell. Biol., 106:2139-2151; Cheng, S-L. et al., 1994, Endo, 134: 277-286; Loffler, G. etal., 1987, Klin. Wochenschr., 65: 812-817; Hauner, H. et al., 1987, J.Clin. Endocrinol. Metabol. 64: 832-835). Differentiation is confirmedusing the histological and immunohistological assays outlined in Table21 and compared to a commercial source of bone marrow-derived MSCs,lineage-specific precursors (positive controls), and human foreskinfibroblasts (HFFs) (negative controls)). The adipose-derived stem cellsare maintained in Control Medium.

TABLE 21 Differentiation Markers And Assays Of Lineage-SpecificDifferentiation. Lineage-specific Histologic/Immunohisto- LineageDeterminant chemical Assay Adipogenic 1. Lipid Accumulation 1. Oil Red Ostain Osteogenic 1. Alkaline phos- 1. Alkaline phosphatase stain phataseactivity 2. Calcified matrix 2. Von Kossa stain productionChondrogenic 1. Sulfated proteogly- 1. Alcian Blue (pH 1.0) staincanrich matrix 2. Safranin O stain 2. Collagen II 3. CollagenII-specific synthesis monoclonal antibody Myogenic 1.Multi-nucleation 1. Phase contrast microscopy 2. Skeletal muscle 2.Myosing and MyoD1 myosin heavy chain specific monoclonal and MyoD1expression antibodies

Adipogenesis

Adipogenic differentiation can be induced by culturing the stem cellsfor 2 weeks in Adipogenic Medium (AM) and assessed using an Oil Red Ostain as an indicator of intracellular lipid accumulation (Preece, A.1972 A Manual for Histologic Technicians, Boston, Mass.: Little, Brown,and Co.). Prior to staining, the cells are fixed for 60 minutes at roomtemperature in 4% formaldehyde/1% calcium and washed with 70% ethanol.The cells are incubated in 2% (w/v) Oil Red O reagent for 5 minutes atroom temperature. Excess stain is removed by washing with 70% ethanol,followed by several changes of distilled water. The cells arecounter-stained for 2 minutes with hematoxylin.

Osteogenesis

Osteogenic differentiation can be induced by culturing the stem cellsfor a minimum of 2 weeks in Osteogenic Medium (OM) and assessed bymeasuring Alkaline Phosphatase (AP) activity and ECM calcification byvon Kossa staining. To detect AP activity, cells are incubated in OM for2 weeks, rinsed with PBS and stained with a 1% AP solution (1% naptholABSI phosphate, 1 mg/ml Fast Red TR) at 37° C. for 30 minutes. For vonKossa staining, the cells are incubated in OM for 4 weeks and fixed with4% paraformaldehyde for 60 minutes at room temperature. The cells arerinsed with distilled water and then overlaid with a 1% (w/v) silvernitrate solution in the absence of light for 30 minutes. The cells arewashed several times with distilled water and developed under UV lightfor 60 minutes. Finally, the cells are counter-stained with 0.1% eosinin ethanol.

Chondrogenesis

Chondrogenic differentiation can be induced using the micromass culturetechnique (Ahrens, P B, et al., 1977 Develop. Biol. 60:69-82; Reddi, A H1982 Prog. Clin. Biol. Res. 110 (part B):261-268; Denker, A E., et al.,1995 Differentiation 59:25-34). Briefly, 10 ml of a concentratedadipose-derived stem cell suspension (8×10⁶ cells/ml) is plated into thecenter of each well and allowed to attach at 37° C. for two hours.Chondrogenic medium (CM) is gently overlaid so as not to detach the cellnodules and cultures are maintained in CM for 2 weeks prior to analysis.Chondrogenesis is confirmed using the histologic stain Alcian Blue atacidic pH. The stem cell nodules are fixed with 4% paraformaldehyde for15 minutes at room temperature and washed with several changes of PBS.Studies have shown specific staining of sulfated proteoglycans, presentin cartilagenous matrices, at pH levels of 1 and below (Lev, R. and S.Spicer 1964 J. Histochem. Cytochem. 12:309-312). Therefore, the cellsare incubated for 30 minutes with 1% (w/v) Alcian Blue (Sigma A-3157) in0.1N HCl (pH 1.0) and washed with 0.1N HCl for 5 minutes to removeexcess stain. In addition to Alcian Blue staining, expression of thecartilage-specific collagen type II isoform also can be determined. Thestem cells are fixed in 4% paraformaldehyde for 15 minutes at roomtemperature. Cells are incubated in 0.2 U/ml chondroitinase ABC for 40min at 37° C. to facilitate antibody access to collagen II. The cellsare rinsed in PBS and endogenous peroxidase activity quenched byincubating for 10 minutes in 3% hydrogen peroxide in methanol. Followinga wash in PBS, non-specific sites are blocked by incubating cells for 1hour in Blocking Buffer (PBS, containing 10% Horse Serum). The cells aresubsequently incubated for 1 hour in Blocking Buffer containing amonoclonal antibody specific to human collagen type II (ICN Biomedical,Costa Mesa, Calif.). The cells are washed extensively in Blocking Bufferand collagen type II visualized using a commercially available kit forthe detection of monoclonal antibodies according to the manufacturer(VectaStain ABC kit, Vector Labs Inc., Burlingame, Calif.).

Myogenesis

Myogenic differentiation can be induced by culturing the adipose-derivedstem cells in Myogenic Medium (MM) for 6 weeks, and can be confirmed byimmunohistochemical staining for the muscle-specific transcriptionfactor, MyoD1 and the myosin heavy chain. Specifically, cells are rinsedtwice with PBS, fixed for 20 minutes with 4% paraformaldehyde and washedseveral times with PBS. The cells are incubated with 3% hydrogenperoxide in PBS for 10 minutes to quench endogenous peroxidase enzymeactivity and nonspecific sites are blocked by incubation in BlockingBuffer (PBS, 10% Horse Serum, 0.1% Triton X-100) for an additional 60minutes. The cells are washed 3 times for 5 minutes each in BlockingBuffer and incubated for 1 hour in Blocking Buffer containing amonoclonal antibody either specific to skeletal muscle myosin heavychain (Biomeda, Foster City, Calif.) or to MyoD1 (Dako Corp,Carpenteria, Calif.). The cells are washed extensively in BlockingBuffer and the monoclonal antibodies visualized using the VectaStain ABCkit according to manufacturer's specifications. The cells arecounterstained with hematoxylin for 3 minutes.

Preparation of Decellularized Adipose Matrix

Decellularized adipose matrix is obtained either using the originalrinseate or the thawed and filtered aspirate obtained during the ASCisolation procedure. Following a series of thorough washes with coldPBS, the washed tissue is soaked in lysis buffer with continuousmechanical agitation. The soaked tissue then is subjected to cell lysisto yield a decellularized tissue.

The sterile decellularized tissue can be subjected to alternateprocedures. For example, (1) it can be lyophilized and milled using afreezer mill to yield a decellularized adipose-derived matrix powder;(2) it can be homogenized to obtain a decellularized adipose-derivedmatrix paste or slurry; (3) it can be homogenized and lyophilized toobtain a three-dimensional adipose-derived matrix; or (4) it can belyophilized to obtain an adipose decellularized tissue matrix sheet.

Recellularization

Adipose-derived decellularized matrices in powder, paste/slurry,three-dimensional or sheet form may be used to reseed isolated ASCs.Following the filtration step for isolating ASCs, the isolatedstromal-vascular fraction (SVF) enriched with ASCs or otherwise purifiedASCs are resuspended in basal or nutrient enriched medium. A portion ofthe resuspended SVF fraction or otherwise purified ASCs are then addedto a sample of an adipose-derived decellularized matrix produced in anyform (powder, paste/slurry, three dimension or sheet). Thedecellularized adipose matrix containing the ASCs is incubated at 37°C.]. The incubation step is followed by static or dynamic seedingconditions for 24 hours, which are well known in the art. There-cellularized adipose matrix is then subjected to a series of cold PBSrinses to wash away unwanted non-adherent cells.

Cryopreservation and Thawing

Additionally, prior to cryopreservation, one or more growth-inductivecomponents optionally can be added. These include, but are not limitedto, bone morphogenic proteins (BMPs), vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), transforming growthfactor beta (TGFβ), platelet-derived growth factor (PDGF), neuralepidermal growth-factor-like 1 (NELL-1), and a combination thereof. Forcryopreservation, for example, mesencult basal media is prepared andsterile filtered. A cryoprotectant solution in basal or nutrient richmedium is added in order to assure full coverage of the tissue.Exemplary cryoprotectant include, but are not limited to, dimethylsulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol,2-Methyl-2.4-pentanediol (MPD), and sucrose. The samples are packaged incryoresistant containers. One sample as a probe sample is cryopreservedin a laboratory cryopreservation unit. The packaged tissue is thensubjected to slow controlled rate freezing to at least −80° C. Once theprogram cycle is complete, the tissue is placed in liquid nitrogen.

Prior to implantation, a cryopreserved adipose implant is thawed. Thethaw procedure warms the tissue preparing it for implantation. The vialcontaining cryopreservation solution and tissue is thawed at roomtemperature or alternatively warmed to 37° C. to expedite the thawingprocess. Alternatively, the thawing temperature can be at a temperaturein the range of about 4° C. through 50° C. Alternatively, thefreeze-thawing process can be repeated. Once the cryopreservationsolution is free flowing, the cryopreservation solution is decanted fromthe vial and the tissue is implanted immediately, without any rinse.Prior to implantation, the tissue is optionally rinsed for 0-15 minuteswith the other wash solutions including but not limited to saline, 5%dextrose in lactated ringers solution, phosphate buffered saline, andany additional isotonic solution.

The wash solution is added at room temperature or alternatively, priorto application to the tissue, the wash solution is warmed to atemperature not exceeding 37° C.-39° C. in order to minimize any damageto the cells contained in the tissue. The wash solution is exchangedthroughout the rinse or alternatively the tissue is stored in the washsolution at 4° C. until ready for implantation. Any remaining tissuefrom the surgery is not be re-frozen for future use. All remainingtissue is disposed off appropriately after surgery.

A strainer is used to contain the tissue during the decanting process.This allows the cryopreservation solution and rinseate to be removedfrom the tissue while minimizing any possible contamination of tissueduring preparation (minimizes human contact). Gauze is optionally usedto contain the tissue during the decant/thaw procedure.

As described in detail above, adipose-derived stem cells possess apotential to differentiate into a wide variety of cell types, including,but not limited to, nerve cells, astrocytes, fat cells, chondrogeniccells, osteogenic cells, or insulin-releasing pancreatic cells.

III. Source Tissue

Placental Tissue

Example 26 Fabrication of Amnion Implant Sheet

Human placentas are recovered from consenting healthy donor mothers atthe time of caesarean section. The amniotic membrane then can beseparated or stripped gently from the underlying chorion layer withminimal damage to the basal membrane. The chorion is optionally used forfabrication of a chorion implant or discarded. A sample of the amnioticmembrane is cut for bioburden assessment. The separated amnionoptionally then is subjected to a series of washes with chilled bufferedisotonic solution to remove unwanted blood debris. This may include aseries of three 5 minute-soaks in a buffered isotonic solution followedby a soak in an antibiotic solution, such as an antibiotic solutioncontaining 1% Penicillin and 1% Streptomycin. The washed amnioticmembrane may then be exposed to a bioburden reducer such as, forexample, surfactants and other cleaning agents to generate preprocessedamnion. The preprocessed amnion may then be subjected to a series ofrinses with cold PBS. The pH of the rinseate is at or near physiologicalpH at the end of the rinse. The preprocessed amnion is then cut intostrips of desired geometry.

Additionally, prior to packaging, amnion sheets may be supplemented withone or more autoinductive components. Examples of autoinductivecomponents include, but are not limited to, growth factors such as BMP-2and 4, VEGF, bFGF, TGF-β, NELL-1, PDGF, and/or a combination thereof.

Additionally, the amnion sheets with or without autoinductivecomponent(s) are placed in a cryopreservation solution. Exemplarycryoprotectants include, but are not limited to, dimethyl sulfoxide(DMSO), glycerol, ethylene glycol, propylene glycol,2-Methyl-2.4-pentanediol (MPD), and sucrose. The samples are packaged incryoresistant containers or packages and cryopreserved using acontrolled rate freezer. Once the program cycle is complete, thecontainers or packages are placed in liquid nitrogen vapor.

Prior to implantation, cryopreserved amnion implant sheets are thawed.The thaw procedure warms the amnion sheets preparing them forimplantation. The package containing cryopreservation solution andamnion sheets is thawed at room temperature or alternatively warmed to37° C. to expedite the thawing process. Once the cryopreservationsolution is free flowing, the cryopreservation solution is decanted fromthe package. Prior to implantation, the amnion sheets are optionallyrinsed for 0-15 minutes with the other wash solutions including, but notlimited to, saline, 5% dextrose in lactated ringers solution, phosphatebuffered saline, and any additional isotonic solution.

The wash solution is added at room temperature or alternatively, priorto application to the tissue, and warmed to a temperature not exceeding37° C.-39° C. in order to minimize any damage to the cells contained inthe amnion sheets. The wash solution is exchanged throughout the rinsesor alternatively the amnion sheets are stored in the wash solution untilready for implantation. Any remaining amnion sheets are not re-frozenfor future use. All remaining unused thawed amnion sheets are disposedoff appropriately after surgery.

A strainer may be used to contain the amnion sheets during the decantingprocess. This allows the cryopreservation solution and rinseate to beremoved from the amnion sheets while minimizing any possiblecontamination of tissue during preparation (minimizes human contact).Gauze is optionally used to contain the tissue during the decant/thawprocedure.

Example 27 Fabrication of Amnion Implant Slurry

Human placentas are recovered from consenting healthy donor mothers atthe time of caesarean section. The amniotic membrane then can beseparated or stripped gently from the underlying chorion layer withminimal damage to the basal membrane. The chorion optionally is used forfabrication of a chorion implant, a combined amnion-chorion implant ordiscarded. A sample of the amniotic membrane is cut for bioburdenassessment. The separated amnion optionally is then subjected to aseries of washes with chilled buffered isotonic solution to removeunwanted blood debris. This may include a series of three 5 minute-soaksin a buffered isotonic solution followed by a soak in an antibioticsolution, such as an antibiotic solution containing 1% Penicillin and 1%Streptomycin. The washed amniotic membrane may then be exposed to abioburden reducer such as, for example, surfactants and other cleaningagents to generate preprocessed amnion. The preprocessed amnion may thenbe subjected to a series of rinses with cold PBS. The pH of the rinseateis at or near physiological pH at the end of the rinse. The preprocessedamnion is then cut into strips of desired geometry.

Alternatively, prior to packaging, the amnion slurry may be supplementedwith one or more autoinductive components. Examples of autoinductivecomponents that can be supplemented to the amnion slurry include, butare not limited to, growth factors such as BMP-2 and 4, VEGF, bFGF,TGF-β, NELL-1, PDGF, and/or a combination thereof.

The amnion slurry with or without autoinductive component(s) is placedin a cryopreservation solution. Exemplary cryoprotectants include, butare not limited to, dimethyl sulfoxide (DMSO), glycerol, ethyleneglycol, propylene glycol, 2-Methyl-2.4-pentanediol (MPD), and sucrose.The samples are packaged in cryoresistant containers and cryopreservedusing a controlled rate freezer. Once the program cycle is complete, thecontainers are placed in liquid nitrogen vapor.

It should be understood by those skilled in the art that manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to improve, forexample, but not limited to, efficiency, and yield.

Prior to implantation, the packages containing cryopreserved amnionimplant slurry are thawed. The thaw procedure warms the amnion slurrypreparing it for implantation. The package containing cryopreservationsolution and amnion slurry is thawed at room temperature oralternatively warmed to 37° C. to expedite the thawing process.Alternatively, the thawing temperature can be at a temperature in therange of about 4° C. through 50° C. Once the cryopreservation solutionis free flowing, the cryopreservation solution is decanted from thepackage. Prior to implantation, the amnion slurry is optionally rinsedfor 0-15 minutes with the other wash solutions including but not limitedto saline, 5% dextrose in lactated ringers solution, phosphate bufferedsaline, and any additional isotonic solution.

The wash solution is added at room temperature or alternatively, priorto application to the slurry, the wash solution is warmed to atemperature not exceeding 37° C.-39° C. in order to minimize any damageto the cells contained in the amnion slurry. The wash solution isexchanged throughout the rinse or alternatively the amnion slurry isstored in the wash solution until ready for implantation. Any remainingamnion slurry is not re-frozen for future use. All remaining unusedthawed portions of amnion slurry are disposed off appropriately aftersurgery.

A strainer may be used to contain the amnion slurry during the decantingprocess. This allows the cryopreservation solution and rinseate to beremoved from the amnion sheets while minimizing any possiblecontamination of tissue during preparation (minimizes human contact). Agauze is optionally used to contain the tissue during the decant/thawprocedure.

Example 28 Fabrication of Amniotic Implant by Reseeding Amniotic StemCells on Decellularized Amniotic Matrix

Human placentas are recovered from consenting healthy donor mothers atthe time of caesarean section. To avoid any potentialblood-transmittable diseases, the pregnant female is prescreened forHIV-1, HIV-2, HTLV-1, hepatitis B and C viruses and syphilis, usingconventional serological tests. Only those placentas for which thematernal blood reveals negative serological results are used to producethe amniotic implant.

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placenta tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into the controlled environment. If the controlledenvironment is a manufacturing hood, the sterile supplies are opened andplaced into the hood using conventional sterile technique. If thecontrolled environment is a clean room, the sterile supplies are openedand placed on a cart covered by a sterile drape. All the work surfacesare covered by a piece of sterile drape using conventional steriletechniques, and the sterile supplies and the processing equipments areplaced on to the sterile drape, again using conventional steriletechniques.

The amniotic membrane can then be separated or stripped gently from theunderlying chorion layer with minimal damage to the basal membrane. Thechorion is optionally used for fabrication of a chorion implant, acombined amnion and chorion implant, or discarded. A sample of theamniotic membrane is cut for bioburden assessment. Optionally, theseparated amnion is then subjected to a series of washes with chilledbuffered isotonic solution to remove unwanted blood debris. This mayinclude a series of three 5 minute-soaks in buffered isotonic solutionfollowed by a soak in an antibiotic solution such as an antibioticsolution containing 1% Penicillin and 1% Streptomycin. The washedamniotic membrane may be then exposed to a bioburden reducer such as,for example, surfactants and other cleaning agents to generatepreprocessed amnion. The amnion sheets may subsequently be subjected toa series of rinses with cold PBS. The pH of the rinseate is at or nearphysiological pH at the end of the rinse. The preprocessed amnion isthen minced using a scalpel or alternatively using scissors to form anamnion slurry. The amnion slurry is then subjected to a series of rinseswith cold PBS. The pH of the rinseate is at or near physiological pH atthe end of the rinse.

Additionally, the processed amnion can be disaggregated mechanicallyand/or treated with digestive enzymes and/or chelating agents thatweaken the connections between neighboring cells, making it possible todisperse the tissue suspension of individual cells.

Isolation of Amniotic Stem Cells

One batch of the rinsed tissue is subjected to a pre-digestion soak for10 minutes in a solution containing trypsin and ethylenediaminetetracetic acid (EDTA). An exemplary trypsin-EDTA solution may contain0.05% Trypsin and 0.5 mM EDTA. The predigestion soak solution isdecanted off and discarded. The presoaked amniotic tissue is subjectedto digestion with 0.05% Trypsin for 40 minutes at 37° C. to yield acrude digest solution. A first portion of the crude digest solution isthen transferred to a sterile tube, neutralized with a small amount ofbasal medium and 10% serum. The resulting neutral digest then iscentrifuged, the supernatant is aspirated and discarded, and the pelletis resuspended in 5 mL of basal medium. The digested tissue of the firstportion is subjected to a subsequent digestion with 0.05% Trypsin for 40minutes at 37° C. to yield a double-digested tissue. Thisdouble-digested tissue is not subjected to neutralization. A secondportion of the crude digest solution is then transferred to a steriletube, neutralized with a small amount of basal medium and [10% serum.The resulting neutral digest is then centrifuged, the supernatantaspirated and discarded and the pellet resuspended in 5 mL of basalmedium to yield a single digested portion. The first neutral digest andthe second neutral digest portions are combined and subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 mL basal media to yield a resuspended solutioncontaining amniotic stem cells.

Additionally, the amniotic epithelial and/or stromal cells may be grownon feeder layers. The use of feeder cells, or an extracellular matrixderived from undifferentiated feeder cells is believed to provide one ormore substances necessary to promote the growth of the stem cells and/orinhibits the rate of differentiation of such cells. Such substances arebelieved to include membrane-bound and/or soluble cell products that aresecreted into the surrounding medium by the cells. For example, amnioticepithelial and/or stromal cells can be grown on a substrate, including,but not limited to, mouse embryo fibroblast cells, STO cells humanfibroblasts, or human epithelium cells and/or combination thereof.Alternatively, additional cell lines can be used with the cell culturemedium to equivalent effect; such additional cell lines can beidentified using standard methods and materials. Alternatively oradditionally, one or more substances produced by the feeder cells, orcontained in the extracellular matrix, can be identified and added tothe cell culture medium of the invention to obviate the need for suchfeeder cells and/or such extracellular matrix.

A sample can be set aside in order to evaluate cell count and cellviability/biological activity of the tissue using commercially availablemethods, including but not limited to, for example, metabolic assays,such as involving luciferase, tetrazolium salts (e.g.,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS),2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3, -4,-5, -8, -9, -10, and -11) and dye exclusion assays such as Tryptan Blue.

In addition, the resuspended solution containing amniotic epithelialand/or stromal cells is placed in a cryopreservation solution. Exemplarycryoprotectants include, but are not limited to, dimethyl sulfoxide(DMSO), glycerol, ethylene glycol, propylene glycol,2-Methyl-2.4-pentanediol (MPD), and sucrose. The samples are packaged incryoresistant containers and cryopreserved using a controlled ratefreezer. Once the program cycle is complete, the containers are placedin liquid nitrogen vapor.

In addition, the isolated aminiotic epithelial and/or stromal cellsexpress antigens, including, but not limited to, CD105+, CD90+, CD73+,CD44+, CD29+, HLA-A, B, C+, CD13+, CD10+, CD166+, CD49d−, CD49e+, CD117(+/− very weak signal), CD14−, CD34−, CD45−, HLA−DR−, and/or acombination thereof.

Alternatively, the isolated amniotic epithelial and/or stromal cellsexpress antigens, including, but not limited to, SSEA-3, SSEA-4+, TRA1-60+, TRA 1-81+, SSEA-1−, and/or a combination thereof.

Alternatively, the isolated aminiotic epithelial and/or stromal expressantigens, including, but not limited to, CD324 (E-cadherin)+, POU5F1+,SOX2+, CFC1+, NANOG+, DPPA3+, PROM1+, PAX6+, FOXD3−, GDF3−, CD140b+,CD349−, GCTM2+, and/or a combination thereof.

Alternatively, the isolated amniotic epithelial and/or stromal cellsexpress antigens, including, but not limited to, Thy-1, OCT-4, SOX2,SSEA3, SSEA4, TRA1-60, TRA1-81, Lefty A, FGF-4, Rex-1 and TDGF-1, and/ora combination thereof.

Studies have shown that amniotic epithelial and/or stromal cells can bedifferentiated into a wide variety of cell types in vitro (Parolini etal, 2008, Stem Cells, 26:300-311). The specific culture conditions fordifferentiation of amniotic stem cells are shown in Table 22:

TABLE 22 Culture conditions for differentiation of amniotic stem cellsDifferentiation Culture Conditions Adipogenic DMEM high glucose (orDMEM/Ham's F-12 medium), 10% FBS, 0.5 mM isobutylmethvlxanthine, 1 μMdexamethasone, 10 μM insulin, 200 μM indomethacin Chondrogenic DMEM highglucose, 1% FBS, 6.25 μg/ml insulin, 10 ng/ml TGF-β1, 50 ng/ml freshascorbic acid Osteogenic DMEM high glucose (or DMEM/Ham's F-12 medium),10% FBS, 10 μM dexamethasone, 10 nM 1-α, 25-dehydroxyvitamin D3, 50μg/ml ascorbic acid, 10 mM β-glycerophosphate MesenCult Human OsteogenicStimulatory Kit (StemCell Technologies) Skeletal DMEM/Ham's F-12 medium(or DMEM high glucose), myogenic 10% FBS, 5% human serum (or horseserum), 50 μM hydrocortisone (0.1 μM dexamethasone) Cardiomyogenic DMEM,10% FBS, 55 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1 mM ascorbicacid 2-phosphate DMEM/Ham's F-12 medium, 10% FBS, 1 mM ascorbic acidNeurogenic DMEM high glucose, 10% FBS, 30 μM all-trans retinoic acidDMEM, 10% FBS, 55 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 5 × 10⁻⁵ Mall-trans retinoic acid, 10 ng/ml FGF-4 DMEM/ Ham's F-12 medium, 10%FCS, 5 × 10⁻⁵ M all-trans retinoic acid, 10 ng/ml FGF4, N-2 supplement(Gibco), B-27 supplement (Gibco) Pancreatic DMEM, 10% FBS, 55 μM2-mercaptoethanol, 1 mM sodium pyruvate, 10 mM nicotinamide on collagenI-coated plate DMEM (or DMEM/Ham's F-12 medium) containing N2 supplement(Gibco), 10 mM nicotinamide Hepatic DMEM, 10% FBS, 55 μM2-mercaptoethanol, 1 mM sodium pyruvate, dexamethansone 10⁻⁷ M, 0.1 μMinsulin for 3 weeks, addition of 1 mM phenobarbital for the final 3days, on collagen I-coated plate DMEM, 10% FBS, 20 ng/ml HGF, 10 ng/mlFGF-2, 10 ng/ml oncostatin M, 100 mM dexamethasone, 10 U/ml heparinsodium salt DMEM/Ham's F-12 medium, 10% FCS + 0.1 μM insulin, 1 × 10⁻⁷ Mdexamethasone Abbreviations: DMEM (Dulbecco's modified Eagle's medium);FBS (fetal bovine serum); FCS (fetal calf serum)

Prior to recellularization, the cryovials containing cryopreservedsuspension of amniotic epithelial and/or stromal cells are thawed. Thecells retain their biological function and viability when thawed. Thethaw procedure warms the cells preparing them for recellularization. Thevial containing cryopreservation solution and amniotic stem cellsuspension is thawed at room temperature or alternatively warmed to 37°C. to expedite the thawing process. Once the cryopreservation solutionis free flowing, an equal volume of a saline solution or alternatively awashing solution is added to the cryopreservation solution to yield anamniotic stem cell suspension. The resulting amniotic stem cellsuspension may be used immediately for recellularization. The thawingprocedure is as described above for example 24.

Alternatively, following preparation of a single cell suspension, theamniotic epithelial and/or stromal cells can be cultured in basalmedium, supplemented with serum, hormones, growth factors, cytokines,antibiotics, trace elements, and other additives. Growth factors thatcan be added include, but are not limited to, fibroblast growth factors(FGFs), epidermal growth factor (EGF), transforming growth factor-β(TGF-β), hepatocyte growth factor (HGF), neural epidermalgrowth-factor-like 1 (NELL-1), or oncostatin M. Additives to the mediummay include insulin, transferrin, selenium, glucose, interleukin-6, andhistone deacetylase inhibitor such as sodium butyrate or tricostatin A.

For example, amniotic epithelial and/or stromal cells are plated ontodishes with DMEM, 10% FBS, 2 mM L-glutamine, EGF (10 ng/ml), insulin (10μg/ml), transferrin (5.5 μg/ml), selenium (6.7 ng/ml) and ethanolamine(2 μg/ml). In addition, sodium pyruvate and non-essential amino acids(1%) may be added to the culture medium. In order to inducedemethylation or dedifferentiation, 5-azacytidine and/or BMP inhibitorscan be added to the medium.

Decellularization

The epithelium layer present on the amnion is substantially removed inorder to expose the basement layer of the amnion. By removing theepithelium, the reduction of an antigenic potential can be expected.Furthermore, since unnecessary cells are removed in advance, target celllayers can be formed. The term “substantially removed” with respect tothe amount of epithelium removed is defined herein as removing greaterthan 90%, greater than 95%, or greater than 99% of the epithelial cellsfrom the amnion. The presence or absence of epithelial cells remainingon the amnion layer can be evaluated using techniques known in the art.For example, after removal of the epithelial cell layer, arepresentative tissue sample from the processing lot is placed onto astandard microscope examination slide. The tissue sample is then stainedusing Eosin Y Stain and evaluated as described below. The sample then iscovered and allowed to stand. Once an adequate amount of time has passedto allow for staining, visual observation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper, the membrane may be frozen, or the epithelial cells may beexposed to nonionic detergents, anionic detergents, and nucleases. Thede-epithelialized tissue then is evaluated to confirm that the basementmembrane has not been compromised and remains intact. For example, arepresentative sample graft is removed for microscopic analysis. Thetissue sample is place onto a standard slide and 100 μl of Eosin Y stainis applied to the sample and allowed to set. The tissue sample then isexamined under magnification. Cellular material will stain darkindicating the presence of cells. If no stained cells are present,de-epithelization has been achieved.

Recellularization

Additionally, amniotic epithelial and/or stromal cells, which are (1)freshly isolated from placenta, (2) thawed from cryopreservation, or (3)cultured in vitro, can be seeded back onto or into decellularizedamniotic tissue matrix by adding a desired number of aminiotic stemcells onto or into decellularized aminiotic matrix and placing thedecellularized aminiotic matrix with the cells into an incubator at 37°C. The cells are allowed to attach to the matrix for up to 24 hoursunder static or dynamic seeding conditions and unwanted, non-adherentcells are rinsed away, if necessary.

Cryopreservation and Thawing

Cryopreservation, storage and thawing procedures for recellularizedamniotic matrix are identical to such procedures described above inexample 24.

As described in detail above, amniotic epithelial and/or stromal cellspossess the potential to differentiate into various cell types,including, but not limited to, adipogenic cells, chondrogenic cells,osteogenic cells, cardiomyogenic cells, neurogenic cells, pancreaticcells, and hepatic cells.

While the described invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the describedinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A composition for repairing a bone defect in apatient, comprising: a cryopreserved mixture prepared without exogenousenzymes and including at least one osteoconductive matrix which includescancellous bone particles; at least one viable population of endogenousosteogenic cells adherent to and resident in an endogenous milieu of atleast some of the cancellous bone particles; and a demineralized bonematrix.
 2. The composition according to claim 1, wherein at least someof the cancellous bone particles have a maximum dimension in a range ofabout 1 mm to about 10 mm.
 3. The composition according to claim 1,wherein at least some of the cancellous bone particles have a maximumdimension no greater than about 4 mm.
 4. The composition according toclaim 1, wherein the cancellous bone particles are allogeneic to thepatient.
 5. The composition according to claim 1, wherein the at leastone viable population of osteogenic cells includes a population ofviable osteoprogenitor cells.
 6. The composition according to claim 1,wherein the demineralized bone matrix includes demineralized corticalbone particles.
 7. The composition according to claim 6, wherein atleast some of the demineralized cortical bone particles have a maximumdimension in a range of about 100 μm to about 1000 μm beforedemineralization.
 8. The composition according to claim 6, wherein atleast some of the demineralized cortical bone particles have elongatedshapes.
 9. The composition according to claim 6, wherein at least someof the demineralized cortical bone particles are in the form of fibers.10. The composition according to claim 6, wherein at least some of thedemineralized cortical bone particles have a maximum dimension in arange of about 1 mm to about 10 mm before demineralization and a minimumdimension in a range of about 5 μm to about 300 μm beforedemineralization.
 11. The composition according to claim 10, wherein atleast some of the demineralized cortical bone particles have elongatedshapes.
 12. The composition according to claim 10, wherein at least someof the demineralized cortical bone particles are in the form of fibers.13. The composition according to claim 6, wherein the demineralizedcortical bone particles are allogeneic to the patient.
 14. Thecomposition according to claim 6, wherein all of the demineralizedcortical bone particles have a residual mineral content of less than0.5% (w/w).
 15. The composition according to claim 1, wherein thecancellous bone particles have a maximum dimension no greater than about4 mm, and the demineralized bone matrix includes demineralized corticalbone particles having a maximum dimension in a range of about 100 μm toabout 1000 μm before demineralization.
 16. The composition according toclaim 1, wherein the cancellous bone particles have a maximum dimensionno greater than about 4 mm, and the demineralized bone matrix includesdemineralized cortical bone particles in the form of fibers having amaximum dimension in a range of about 1 mm to about 10 mm beforedemineralization and a minimum dimension in a range of about 5 μm toabout 300 μm before demineralization.
 17. The composition according toclaim 6, wherein at least some of the demineralized cortical boneparticles have a maximum dimension in a range of about 10 μm to about 20cm before demineralization and a minimum dimension in a range of about 5μm to about 300 μm before demineralization.
 18. The compositionaccording to claim 17, wherein at least some of the demineralizedcortical bone particles have elongated shapes.
 19. The compositionaccording to claim 17, wherein at least some of the demineralizedcortical bone particles are in the form of fibers.
 20. The compositionaccording to claim 1, wherein the cancellous bone particles have amaximum dimension no greater than about 4 mm, the endogenous osteogeniccells are resident in at least some of the cancellous bone particles,and the demineralized bone matrix includes demineralized cortical boneparticles in the form of fibers having a maximum dimension in a range ofabout 10 μm to about 20 cm before demineralization and a minimumdimension in a range of about 5 μm to about 300 μm beforedemineralization.
 21. The composition according to claim 1, wherein theat least one osteoconductive matrix does not include measurableimmunologically reactive hematopoetic cells.
 22. The compositionaccording to claim 1, wherein the cancellous bone particles do notinclude measurable immunologically reactive hematopoetic cells.
 23. Thecomposition according to claim 1, wherein the cancellous bone particlesand the demineralized bone matrix are allogeneic to the patient, and thecomposition does not initiate an immune reaction after implantation in apatient.
 24. The composition according to claim 1, wherein the at leastone viable population of endogenous osteogenic cells adherent to andresident in the endogenous milieu of the growth conductive matrix isimmune privileged.
 25. The composition according to claim 1, wherein themixture does not include cells exogenous to the at least oneosteoconductive matrix and the demineralized bone matrix.
 26. Thecomposition of claim 1, wherein the mixture further comprises a carrier.27. The composition of claim 26, wherein the carrier includes acomponent selected from the group consisting of an isotonic solution, asodium chloride solution, a lactated Ringer's solution,phosphate-buffered saline, platelet rich plasma, a hyaluronic acid, ahyaluronic acid derivative, a hyaluronate salt, a hyaluronate ester,thrombin, fibrin, glycerin, collagen, lecithin, and a combinationthereof.
 28. The composition of claim 1, wherein the mixture furthercomprises a polysaccharide.
 29. The composition of claim 1, wherein themixture further comprises a glycosaminoglycan.
 30. The composition ofclaim 1, wherein the mixture further comprises a biological componentthat is not bone.
 31. The composition of claim 30, wherein thebiological component that is not bone is selected from the groupconsisting of a DNA, an RNA, a short hairpin RNA, a small interferingRNA, micro RNA, a polysaccharide, a peptide, a matrix protein, aglycosaminoglycan, a viral vector, a liposome, and a combinationthereof.
 32. A tissue-derived implant for repairing a bone defect in apatient, comprising a cryopreserved mixture prepared without exogenousenzymes and including an extracellular matrix which is osteoinductiveand/or osteoconductive; and at least one viable population of osteogeniccells, wherein the osteogenic cells include one or more of an osteogenicstem cell, a tissuegenic cell, a precursor cell, a progenitor cell, animmature cell, a non-terminally differentiated cell, a cell withdifferentiation potential, or a combination thereof.
 33. Thetissue-derived implant of claim 32, wherein the extracellular matrixincludes demineralized osteoinductive bone particles.
 34. Thetissue-derived implant of claim 32, wherein the extracellular matrixincludes non-demineralized osteoconductive bone particles.
 35. Thetissue-derived implant of claim 32, wherein the mixture furthercomprises a carrier.
 36. The tissue-derived implant of claim 32, whereinthe mixture further comprises a polysaccharide.
 37. The tissue-derivedimplant of claim 32, wherein the mixture further comprises aglycosaminoglycan.
 38. The tissue-derived implant of claim 32, whereinthe mixture further comprises a biological component that is not bone.39. The tissue-derived implant of claim 32, wherein the mixture isprepared without exogenous enzymes.
 40. A tissue-derived implant forimplantation in a patient allogeneic to a donor of the implant, theimplant as a whole comprising: a cryopreserved mixture prepared withoutexogenous enzymes and including (a) at least one tissue-derivedgrowth-conductive matrix of the donor from which measurableimmunologically-reactive hematopoietic cells have been removed; and (b)at least one viable population of non-expanded cells endogenous to thegrowth-conductive matrix of the donor that remains adherent to andresident in an endogenous milieu of the growth-conductive matrix, theviable population of cells including one or more of a tissuegenic cell,a precursor cell, a progenitor cell, an immature cell, a non-terminallydifferentiated cell, a cell with differentiation potential and acombination thereof; with the proviso that, before implantation, themixture is not reconstituted with cells exogenous to the tissue-derivedgrowth-conductive matrix of the donor.
 41. The tissue-derived implant ofclaim 40, wherein the mixture is prepared without exogenous enzymes.